|
|
|
|
|
| J Gen Microbiol, 1985 May, 131 ( Pt 5), 1130 - 40 Plasmid fusions mediated by one end of TnA; Heritage J et al.; We have observed plasmid fusions in a recA background mediated by a single end of TnA . These occur when transposase is provided either in cis or in trans . Insertions of the plasmid carrying the TnA inverted repeat sequence occur at many sites in the target plasmid . The point of fusion on the plasmid carrying TnA sequences always appears to be located in the region which carries the TnA inverted repeat sequence . In contrast to the transposition of an intact TnA element, plasmid fusions mediated by one end of TnA are very rare events . The implications of our results for models of transposition are discussed. J Gen Microbiol, 1985 May, 131 ( Pt 5), 1123 - 30 Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161; Meyer JF et al.; The isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described . A 1.7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a cointegrate structure that carries two direct repeated copies of the IS element . By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene. Genetika, 1985 May, 21(5), 748 - 55 {Physical and genetic organization of the plasmid R15}; Dobritsa AP et al.; The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively . The restriction sites were located on the physical map of the R15 genome . Distribution of the cleavage sites is strongly asymmetric . 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354 . According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation . The features of physical and genetic structures of R15 and other IncN plasmids are discussed. Antimicrob Agents Chemother, 1985 May, 27(5), 879 - 81 Resistance to various tetracyclines mediated by transposon Tn10 in Escherichia coli K-12; Traub B et al.; Levels of resistance to tetracycline, chlortetracycline, demethylchlortetracycline, doxycycline, oxytetracycline, methacycline, pyrrolidinotetracycline, minocycline, and beta-chelocardin of Escherichia coli K-12 carrying transposon Tn10 or defined DNA segments of Tn10 were determined . In all cases, tetA was the only gene required for resistance . Doxycycline was the most effective inducer of tetA gene expression. Tsitologiia, 1985 May, 27(5), 565 - 71 {Genetic transformation of somatic cells . III . An analysis of the status of the plasmid nucleotide sequences in the extrachromosomal DNA of transformant clone cells and the rescue of extrachromosomal molecules of the plasmid DNA}; Tomilin NV et al.; Extrachromosomal DNAs from TK+ transformant clones of A238 Chinese hamster cells isolated after the treatment with plasmid pST826 containing thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV1) and 1.8 kb insert of human satellite III DNA (HSIII) were studied by hybridization technique . In two TK+-clones (2T301 and 2T16) large quantities of rearranged plasmid DNA molecules were found . Electron microscopy show in clone 2T301 the presence of circular DNAs with average length being 4.64 +/- 0.27 kb . These molecules were rescued by retransformation into E . coli and analysed by restriction mapping and hybridization . All of them contain deletions spanning the entire TK gene of HSV1 and pBR325 sequences situated just downstream from the ORI of replication . The origin of extra-replicating circular DNA in 2T301 clone is discussed. Tsitologiia, 1985 May, 27(5), 554 - 64 {Genetic transformation of somatic cells . II . An analysis of the status of the plasmid nucleotide sequences in chromosomal DNA and the thymidine kinase activity in transformant clone cells}; Tomilin NV et al.; Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1) . The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones . These sequences are probably tandemly arranged, and each cluster contains 25--50 copies . SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments . Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9) . The results suggest that the true TK+-transformant cells are selected by the procedure used in this study. Prostaglandins, 1985 May, 29(5), 703 - 13 Effect of the thromboxane receptor antagonist EP 092 on endotoxin shock in the sheep; Armstrong RA et al.; The thromboxane receptor antagonist EP 092 inhibits the acute pulmonary vascular response to E . coli endotoxin in the anaesthetized, closed-chest sheep . The increase in the TXB2 level in arterial blood was not suppressed by EP 092 . Intravenous infusion of the thromboxane mimetic 11,9-epoxymethano PGH2, but not PGF2 alpha, raises pulmonary artery pressure and lowers arterial pO2 similar to the endotoxin . Isolated strips of lobar pulmonary veins but not lobar arteries are contracted by low concentrations of 11,9-epoxymethano PGH2 - the effects are potently inhibited by EP 092. EMBO J, 1985 May, 4(5), 1351 - 5 Evidence for a repeating domain in type I restriction enzymes; Argos P; The primary structures of the recognition subunit (hsdS) in type I restriction enzymes from three isolates of Escherichia coli were compared and aligned by use of amino acid physical properties . A repeating domain was found in each of the subunits suggesting a pseudo-dimeric structure . Secondary structure predictions delineated two helical regions in each domain which suggested that the recognition subunits may act in a fashion similar to that proposed for repressor and activator molecules; namely, interaction with double-stranded DNA through helices and in two successive major grooves on the same DNA side . One helical motif could provide the specific recognition site and the other, the restriction site. Biochem J, 1985 May 1, 227(3), 925 - 31 Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli; George GN et al.; The interconversion of nitrate reductase from Escherichia coli between low-pH and high-pH Mo(V) e.p.r . signal-giving species was re-investigated {cf . Vincent & Bray (1978) Biochem . J . 171, 639-647} . The process cannot be described by a single pK value, since the apparent pK for interconversion is raised by the presence of various anions . The low-pH form of the enzyme exists as a series of complexes with different anion ligands of molybdenum . Each complex has specific and slightly different e.p.r . parameters, but all show strong coupling of Mo(V) to a single proton, exchangeable with the solvent, having A(1H)av . 1.0 to 1.3 mT . Complexes with Cl-, F- {A(19F)av . 0.7 mT}, NO3- and NO2- give particularly well-defined spectra . The high-pH form of the enzyme is now shown to bear a coupled proton . Like that in the low-pH species, this proton is exchangeable with the solvent, but the coupling is much weaker, with A(1H)av . 0.3 mT . Thus, contrary to earlier assumptions, the proton detectable by e.p.r . is probably not identical with the proton whose dissociation controls interconversion between the two species; the latter proton could be located in the protein rather than on a ligand of molybdenum . Treatment of the enzyme with trypsin {Morpeth & Boxer (1985) Biochemistry 24, 40-46} did not affect its Mo(V) e.p.r . signals. Proc Natl Acad Sci U S A, 1985 May, 82(10), 3242 - 6 Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa; Bryant DA et al.; The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis . The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe . Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment . Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment . This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning . The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined . The two open reading frames are in the same orientation and are separated by 39 base pairs . AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence . Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes . A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation . The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome. Exp Cell Res, 1985 May, 158(1), 237 - 43 Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture; Bernard HU et al.; Mouse L fibroblasts and other mammalian cells are killed by the translation inhibitor hygromycin B . We have modified the gene conferring resistance against hygromycin B in E . coli in such a way that it can be transcribed in mammalian cells from the promoter of the HSVtk gene . The resulting plasmid, pHMR272, was transfected into mouse L fibroblasts and HeLa cells by the calcium phosphate method and upon selection produced clones resistant against hygromycin B . The transfection rate was similar to that obtained with other selective markers . This plasmid is a useful addition to the relatively small number of dominant selectable markers available for mammalian cells. Proc Natl Acad Sci U S A, 1985 May, 82(9), 3015 - 9 Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli; Krause DC et al.; Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator . Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with {alpha-32P}ATP in 100 mM sodium phosphate buffer . This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations . Transformation studies with purified pMW286 established that the ability of E . coli cells to accumulate radioactivity was mediated by the recombinant plasmid . Results from experiments in which {3H}ATP was substituted for {alpha-32P}ATP strongly suggested that the radiolabeled ATP was transported intact . Furthermore, {3H}ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner . Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP . Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides . The successful cloning and expression of the rickettsial ADP/ATP translocator in E . coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism. Proc Natl Acad Sci U S A, 1985 May, 82(9), 2570 - 3 Direct measurement of the electrogenic activity of o-type cytochrome oxidase from Escherichia coli reconstituted into planar lipid bilayers; Hamamoto T et al.; Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by means of vectorial electron flow . In the experiments reported here, purified oxidase is reconstituted in planar lipid bilayers formed at the tip of patch pipets, and open-circuit membrane potentials generated by electron transfer are measured directly . Potentials of up to 4 mV (substrate side positive) are generated in the presence of reduced phenazine methosulfate or ubiquinol-1, and with both substrates, electrogenic activity is inhibited by cyanide . Furthermore, the membrane potential generated during oxidase turnover is inhibited progressively with applied voltages (substrate side positive), decreasing almost to zero at an applied voltage of 150 mV. Mutat Res, 1985 May, 149(3), 303 - 10 An assessment of the importance of error-prone repair and point mutations to forward mutation to L-azetidine-2-carboxylic acid resistance in Escherichia coli; Mitchell ID et al.; By comparison of E . coli WP2 with CM891 (uvrA- pKM101) we found that pKM101 plasmid and uvrA- mutation considerably enhanced both spontaneous and chemically-induced reversion at the trp locus . However, little or no increase was observed for forward mutation at the A2C locus . Furthermore, mutation frequency decline was considerably greater for trp reversion than for mutation to A2Cr . Thus neither error-prone repair nor point mutation seemed likely to be the major mechanism for forward mutation at the A2C locus . Results for spontaneous mutation of recA-, polA- and gyrA- strains showed that polA- and gyrA- gave good increases in forward mutation but not in reversion . It was inferred that deletion, transposition and/or larger chromosomal effects rather than point mutation were mainly responsible for most forward mutation. Mutat Res, 1985 May, 149(3), 297 - 302 A loss of uvrA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli; Yamamoto K et al.; We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis . Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau . The induction was blocked by recA and lexA(Ind-) mutations that conferred no mutagenesis on the cell . Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C . The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants. J Virol, 1985 May, 54(2), 509 - 14 Herpes simplex virus 1 reiterated S component sequences (c1) situated between the a sequence and alpha 4 gene are not essential for virus replication; Hubenthal-Voss J et al.; The herpes simplex virus 1 genome consists of two components, L and S, each containing unique sequences flanked by inverted repeats . Each of the 6.5-kilobase pair inverted repeats of the S component, designated a'c' and ca, contains an approximately 700-base pair sequence (designated c1) located between the a sequence and the 3' terminus of the alpha 4 gene . Like the a sequence, c1 consists of direct repeats and unique sequences . Its function is not known . To probe for its function, we constructed a plasmid containing a viral thymidine kinase (TK) gene inserted into the c1 sequence . The construct was recombined into the genome of a TK- virus by cotransfection with intact viral DNA and selection for TK+ virus . As predicted from previous studies (Knipe et al., Proc . Natl . Acad . Sci . U.S.A . 75:3896-3900, 1978), the TK gene was found to be present in both copies of the c1 sequence in the R3104 virus . To delete the c1 sequence we constructed a plasmid containing 4 kilobase pairs of pBR322 flanked by an a sequence and by structural sequences of the alpha 4 gene . In this instance the cells were transfected with the construct and R3104 DNA; the progeny of the transfection was plated in the presence of 5-bromo-2'-deoxyuridine, and the selection was for TK- virus (R3158) . The pBR322 DNA sequences replaced the c1 at both termini of the S component in R3158 DNA, but a sequence homologous to c1 was present in proximity to the 3' terminus of the alpha 4 gene . The results indicate that the c1 region has no significant role in the replication of the virus in cell culture . The advantage of inserting the pBR322 sequence is that it permits efficient cloning of large herpes simplex virus 1 DNA fragments by simple ligation of digests and transformation of appropriate Escherichia coli strains . The effortless selection of recombinants carrying inserts in both copies of the c1 restates the usefulness of this technique for selection of insertion deletion recombinants and underscores the rapid emergence of sequence identity at both ends of the reiterated regions of the S component as previously reported (Knipe et al., Proc . Natl . Acad . Sci . U.S.A . 75:3896-3900, 1978). J Bacteriol, 1985 May, 162(2), 855 - 7 Preferential inhibition of plasmid replication in vivo by altered DNA gyrase activity in Escherichia coli; Uhlin BE et al.; The thermosensitive growth phenotype exerted by runaway-mutant plasmids was suppressed by sublethal doses of the DNA gyrase inhibitors novobiocin or nalidixic acid, although the latter drug was less efficient . A novobiocin-resistant gyrB mutant Escherichia coli strain prevented expression of the runaway phenotype at 37 to 42 degrees C in the absence of any drug. J Bacteriol, 1985 May, 162(2), 615 - 20 Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene; Lee CH et al.; The Escherichia coli heat-stable enterotoxin STII gene in P307 is flanked by inverted repeat sequences, suggesting that the STII gene is part of a transposon . To study the transposability, a DNA fragment containing the putative STII transposon has been cloned . Results of transposition assays indicated that the STII gene can transpose from one plasmid to another . The size of the transposon has been determined to be approximately 9 kilobases . The structure and the location of the STII gene in clinical isolates of Escherichia coli have been investigated by restriction enzyme analyses . The structural genes of STII from different clinical isolates appear to be uniform in size, but the flanking sequences are heterogeneous . This result suggests that the STII genes in different isolates are not on the same transposon as observed in P307. J Bacteriol, 1985 May, 162(2), 529 - 34 Recombination in recA cells between direct repeats of insertion element IS1; Braedt G; The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain . The recombination requires the lambda pL promoter on the plasmid . A plasmid that contains mutant IS1 elements does not recombine . These results indicate that this recombination requires an IS1-specific gene product . The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1 . I discuss a possible role for the lambda pL promoter. Cancer Genet Cytogenet, 1985 May, 17(1), 69 - 74 Cytogenetic studies of stimulated lymphocytes in hairy cell leukemia; Sadamori N et al.; Using a sister chromatid differentiation (SCD) technique, cell cycle analysis in lymphocytes from two patients with hairy cell leukemia (HCL) revealed it to be similar to cell cycle progression of normal lymphocytes stimulated with lipopolysaccharide W from Escherichia coli 0.55:B5 (LPS) . It appears that LPS can readily stimulate the leukemic cells of HCL into mitosis . In the two cases of B cell HCL studied, one (case 1) was revealed to have an abnormal clone with a missing chromosome #22 that was related to the production of lambda-chains. Mutat Res, 1985 May, 145(3), 107 - 12 Role of the umuC gene in postreplication repair in UV-irradiated Escherichia coli K-12 uvrB; Wang TC et al.; The role of the umuC gene product in postreplication repair was studied in UV-irradiated Escherichia coli K-12 uvrB cells . A mutation at umuC increased the UV radiation sensitivities of uvrB, uvrB recF, uvrB recB, and uvrB recF recB cells; it also increased the deficiencies in the repair of DNA daughter-strand gaps in these strains, but it did not affect the repair of DNA double-strand breaks that arose from unrepaired DNA daughter-strand gaps . We suggest that the umuC gene product is involved in a minor system for the repair of DNA daughter-strand gaps, possibly the repair of overlapping DNA daughter-strand gaps. J Ultrastruct Res, 1985 May, 91(2), 138 - 48 Sputter shadowing improved by using a tungsten target; Colquhoun WR et al.; This work builds upon a previous paper (W . Colquhoun, 1984, J . Ultrastruct . Res . 87, 97) in which a sputter shadowing device was briefly described . The device allowed TEM specimens to be shadowed in a conventional sputter coater . Images obtained by sputter shadowing with a standard Au/Pd target were of good quality but were slightly inferior to the best that could be obtained by e--beam evaporation of tungsten . Here we show that construction and use of a tungsten target greatly improves the quality of the sputter shadowed deposit . Images of DNA and ribosomal subunits contrasted by sputter shadowing with tungsten are shown . The DNA images indicate that sputter shadowing with tungsten is a gentle contrasting technique . The sputter shadowed images of the 30 S ribosomal subunits show the major features of the particle revealed by evaporation shadowing using the most sophisticated of methods in that technology . Advantages of sputter shadowing are discussed and a rationale for the improved grain obtained by sputtering tungsten is suggested. Virology, 1985 May, 143(1), 352 - 6 Plating efficiencies of modified lambda bio particles on temperature-sensitive hsd mutants of Escherichia coli K12; Fuerst CR; Two mutants of Escherichia coli K12 that are temperature sensitive in cell growth and lambda phage production are shown to contain at least two mutations . One of the mutations in each of the isolates is in the hsd locus, and modification and restriction of lambda exhibits temperature sensitivity . One of the hsd mutations causes plaque formation by modified lambda bio particles that do not contain an intact ral gene to be temperature dependent. J Biochem (Tokyo), 1985 May, 97(5), 1401 - 7 Change of inhibitor sensitivities of Escherichia coli F1-ATPase due to a mutational substitution of Phe for Ser at residue 174 of the beta subunit; Takeda K et al.; The F1-ATPase from the uncD11 mutant of E . coli (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., & Futai, M . (1980) J . Biochem . 88, 695-703), showed different enzymological properties from the wild-type enzyme . The mutant F1-ATPase had biphasic kinetics and essentially the same Km values as the wild-type enzyme, although its Vmax values were lower . The mutant enzyme showed altered sensitivities to dicyclohexylcarbodiimide (DCCD), azide and quercetin; it was less sensitive than the wild-type to quercetin and DCCD, and its Mg2+-dependent ATPase activity was slightly more resistant to azide than that of the wild-type, whereas its Ca2+-dependent activity was more sensitive . On the other hand, the mutant and wild-type F1 were inhibited equally by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) . The fact that the Mg2+- and Ca2+-dependent F1-ATPase activities of the wild-type and mutant responded differently to quercetin and azide suggested that their mechanisms of action were different . Previous studies (Noumi, T., Mosher, M.E., Natori, S., Futai, M., & Kanazawa, H . (1984) J . Biol . Chem . 259, 10071-10075) indicated that Ser is replaced by Phe at residue 174 of the beta subunit of the mutant . Thus the Ser residue or its neighboring area(s) may constitute the binding site of DCCD, quercetin and azide. Infection, 1985 May-Jun, 13(3), 159 - 62 P-fimbriae studies on the diagnosis and prevention of acute pyelonephritis; Kallenius G et al.; Theoretically there are several ways to prevent pyelonephritis and renal scarring caused by P-fimbriated Escherichia coli . Screening for individuals at risk, e.g . those carrying P-fimbriated pyelonephritogenic E . coli or those with high receptor density on their uroepithelial cells, could perhaps define a population where prophylaxis with a receptor analogue or vaccination with P-fimbriae may be relevant . Epidemiological measures in neonatal and maternity wards may prevent the nosocomial spread of virulent bacteria and reduce the number of colonized infants . However, no such methods have so far had any proven clinical relevance, and today, the important concern is still to try by conventional means--as we have always done--to get an early diagnosis and to treat the patient without delay. J Gen Microbiol, 1985 May, 131 ( Pt 5), 1263 - 6 Organization of fimbriate cells in colonies of Escherichia coli strain 3040; Nowicki B et al.; Immunofluorescence staining with fimbria-specific antibodies was used to study the organization of fimbriate cells in colonies of Escherichia coli strain 3040 . The strain has both type-1 and S fimbriae and shows fast phase variation between the fimbrial types . Colonies stained in sectors whose length and number per colony were dependent on the fimbrial phase of progeny cells . It is proposed that such sectors result from fimbrial phase variation. J Gen Microbiol, 1985 May, 131 ( Pt 5), 1115 - 21 Phage pilH alpha: a phage which adsorbs to IncHI and IncHII plasmid-coded pili; Coetzee JN et al.; Phage pilH alpha was specific for bacterial strains, of various genera, harbouring plasmids of the HI and HII incompatibility groups . Plaque formation was temperature sensitive in that plaques formed at 26 degrees C but not at 37 degrees C . Plaques were fairly clear, irregular in outline and varied from pin point to about 2 mm in diameter on all hosts where plaques were detected . The phage had an isometric hexagonal outline with a diameter of 25 nm . It contained RNA but differed from all but one other plasmid-dependent RNA phage by being sensitive to chloroform . It adsorbed along the length of the shafts of IncHI and HII plasmid-coded pili. J Bacteriol, 1985 May, 162(2), 668 - 75 Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli; Freitag CS et al.; Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-) . Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead . After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region . Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting . When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage . All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim . Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction. Infect Immun, 1985 May, 48(2), 378 - 83 Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli; Nataro JP et al.; Histopathological evidence suggests that the adherence of enteropathogenic Escherichia coli (EPEC) to the mucosa of the small bowel is an important step in pathogenesis . Several reports have shown that many EPEC isolates adhere to HEp-2 and HeLa cells in tissue cultures . In the HeLa cell assay, there are at least two distinct patterns of adherence: localized adherence, which is characterized by the formation of bacterial microcolonies, and diffuse adherence, in which bacteria cover the cell uniformly . We have found that these two patterns can be demonstrated in HEp-2 cells as well as in HeLa cells and that the results of the two assays are closely correlated . Using a DNA probe which is sensitive and specific for localized adherence to HEp-2 cells, we provide evidence that localized adherence and diffuse adherence by EPEC are due to at least two genetically distinct adhesions which confer phenotypic differences in both the morphology of HEp-2 cell adherence and in surface hydrophobicity . The two factors are each encoded on plasmids which vary in size from 55 to 70 megadaltons; one strain exhibiting localized adherence carried these genes on the chromosome. Infect Immun, 1985 May, 48(2), 318 - 23 Binding of fibronectin to human buccal epithelial cells inhibits the binding of type 1 fimbriated Escherichia coli; Simpson WA et al.; The interaction of purified human plasma fibronectin (FN) with human buccal epithelial cells was studied . Maximal binding of FN occurred at pH 5 . The majority of the binding was specific and reversible . The binding of FN to buccal cells was saturable, reaching a maximum when 10(5) buccal cells were incubated with approximately 200 micrograms of radiolabeled protein per ml . The adherence of a type 1 fimbriated strain of Escherichia coli to buccal epithelial cells was inhibited by the addition of FN in a dose-related manner . Our results indicate that exogenous FN can bind to human buccal epithelial cells and block the attachment of a type 1 fimbriated strain of E . coli. Proc Natl Acad Sci U S A, 1985 May, 82(10), 3182 - 6 Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis; Povirk LF et al.; When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues . In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used . Chromophore-induced release of free cytosine base from {3H}cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols . These results suggest that the alkali-dependent strand break is some form of apyrimidinic site . These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA . These sites were also less sensitive to E . coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E . coli endonuclease III by a factor of at least 10 . These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites . When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites . All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites . The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin. Proc Natl Acad Sci U S A, 1985 May, 82(9), 2829 - 33 Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression; Clare J et al.; We have determined the DNA sequence of the transposable element Ty912 of yeast . The 5918-base-pair element encodes two genes, tya912 and tyb912, which specify proteins similar to sequence-specific DNA-binding proteins of Escherichia coli and retroviral reverse transcriptases, respectively . The tyb912 gene is atypical of eukaryotic genes since (i) it begins 1336 nucleotides into the Ty912 mRNA (i.e., downstream of the tya912 gene) and (ii) the first in-frame AUG is 921 nucleotides into the coding frame . Protein blot analysis of Ty-lacZ fusions shows that the tyb912 gene is translated starting at the 5' end of the tya912 gene and that the primary translational product is a tya912::tyb912 fusion protein . We have shown that synthesis of this fusion protein probably does not occur by RNA splicing . The data are consistent with a mechanism of translational frameshifting occurring within the region of overlap between the 3' end of tya912 and the 5' end of tyb912. Proc Natl Acad Sci U S A, 1985 May, 82(9), 2583 - 7 Dissection of Mycobacterium tuberculosis antigens using recombinant DNA; Young RA et al.; A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies . M . tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library . Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome . Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes . This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M . tuberculosis . The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Mutat Res, 1985 May, 149(3), 287 - 96 Involvement of DNA lesions and SOS functions in 5-bromouracil-induced mutagenesis; Pietrzykowska I et al.; Mutagenesis resulting from incorporation of 5-bromouracil (BU) in the DNA of E . coli K12 proceeds largely (approximately 80%) via misrepair of the lesions resulting from incorporation of the analogue . The premutational lesions are due principally to dehalogenation of incorporated BU residues, leading to formation of uracil residues, and removal of these by uracil-DNA glycosylase with formation of apyrimidinic sites . In the xthA mutant, defective in AP endonuclease, there is a several-fold increase in the frequency of BU-induced mutations, underlining the importance of AP sites in BU-induced mutagenesis . Premutational lesions undergo mutation frequency decline (MFD), which is subject to delay in the xthA mutant, pointing to some role of AP endonuclease in MFD, and further supporting involvement of AP sites in BU-induced mutagenesis . Efficient BU mutagenesis is dependent on the functions of the genes recA and umuC and non-mutated lexA protein. Infect Immun, 1985 May, 48(2), 372 - 7 Partial amino acid sequence and molecular cloning of the encoding gene for the major outer membrane protein of Chlamydia trachomatis; Nano FE et al.; The first 25 N-terminal amino acids of the major outer membrane protein of Chlamydia trachomatis serovar L2 were determined . The amino acid sequence was used to construct an oligonucleotide probe specific for the major outer membrane protein gene . Using this oligonucleotide as a hybridization major outer membrane protein gene . Using this oligonucleotide as a hybridization probe, we discovered one recombinant clone that produced a 15-kilodalton polypeptide which reacted with a monoclonal antibody directed against the major outer membrane protein type-specific epitope . In a separate set of experiments, we uncovered another recombinant clone that produced a 51-kilodalton polypeptide which was reactive with an anti-major outer membrane protein subspecies-specific monoclonal antibody . The expression of these recombinant DNA plasmids in Escherichia coli is discussed. Infect Immun, 1985 May, 48(2), 350 - 4 Role of Escherichia coli type 1 pilus in colonization of porcine ileum and its protective nature as a vaccine antigen in controlling colibacillosis; Jayappa HG et al.; This study was designed to evaluate the role of Escherichia coli type 1 pili in adherence of the organism to porcine small intestines and the efficacy of pili as a vaccine antigen in controlling neonatal colibacillosis . Our results demonstrated that an E . coli phase cloned to express type 1 pili readily attached to the small intestines of colostrum-deprived newborn pigs . Immunofluorescent staining of intestine sections revealed the presence of E . coli expressing type 1 pili only on the brush border, suggesting involvement of type 1 pili in the colonization process . Administration of anti-type 1 serum to newborn pigs prior to challenge reduced the level of gut-associated E . coli sixfold compared with controls . Purified type 1 pilus vaccine induced significant protection against colibacillosis in newborn pigs following challenge with E . coli expressing type 1 pili . Pigs born to vaccinated gilts scoured less and gained more weight than pigs born to control gilts . Our results demonstrate that type 1 pili are a virulence factor, as well as an effective vaccine antigen. Mikrobiologiia, 1985 May-Jun, 54(3), 398 - 401 {Effect of inhibitors on the viability and protein and nucleic acid synthesis of Escherichia coli}; Avtushenko SS et al.; The object of this work was to study how the synthesis of protein, RNA and DNA in Escherichia coli M17 and its viability were influenced by chloramphenicol (50 and 300 micrograms/ml) an inhibitor of protein biosynthesis, and sodium azide (200 and 2000 microM) and aminazine (50 micrograms/ml), inhibitors of respiration . The exposed were inhibitors with the bacteria for 60 min at room temperature and for 1-4 months at -10 degrees C . The inhibition of the E . coli viability by chloramphenicol was shown to be reversible . The respiration inhibitors stabilized its viability upon storage at -10 degrees C for one month . The inhibitors were found to produce a different effect on the synthesis of RNA and protein in E . coli . The rates of DNA synthesis hardly changed . No correlation was established between changes in the synthesis of protein and nucleic acids by E . coli after the action of the inhibitors and its viability. Mol Biol (Mosk), 1985 May-Jun, 19(3), 791 - 9 {Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon}; Shpaer EG; The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli . It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon . The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed . The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes . For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001) . These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E . coli. Mol Biol (Mosk), 1985 May-Jun, 19(3), 702 - 16 {The Shine-Dalgarno sequence and the effectiveness of translation initiation}; Khudiakov IuE; On the basis of theoretical analysis of different mRNAs secondary structure it is suggested that the efficiency of procaryotic translation initiation depends to a great extent on the possibility to generate a single-stranded region around the initiation codon . The local disruption of the mRNA secondary structure is mostly determined by interaction according to Shine--Dalgarno of 16S rRNA with the complementary mRNA region . Other mechanisms of single-stranded region generation in the initiation zone of mRNA are discussed. Mol Biol (Mosk), 1985 May-Jun, 19(3), 597 - 609 {The context analysis of polynucleotide sequences . II . Inverted repeats and complementary palindromes in RNA-polymerase genes}; Zharkikh AA et al.; A new approach to the reconstruction of the RNA secondary structure is suggested on the basis of the method of contextual analysis of polynucleotide sequences . The coding gene regions of beta-, beta'-, sigma-subunits of E . coli RNA polymerase and of phage T7 RNA polymerase were analysed . The clusters of non-random inverted repeats were found in all these genes . The mRNA coded by them can be folded into compact secondary structures . The latter are formed by quite long helices with a few cases of mispairing. Biochimie, 1985 May, 67(5), 517 - 21 {Extraction of symbolic determinants common to a family of biological sequences}; Saurin W et al.; A set of sequences can be defined by their common subsequences, and the length of these is a measure of the overall resemblance of the set . Each subsequence corresponds to a succession of symbols embedded in every sequence, following the same order but not necessarily contiguous . Determining the longest common subsequence (LCS) requires the exhaustive testing of all possible common subsequences, which sum up to about 2L, if L is the length of the shortest sequence . We present a polynomial algorithm (O(n X L4), where n is the number of sequences) for generating strings related to the LCS and constructed with the sequence alphabet and an indetermination symbol . Such strings are iteratively improved by deleting indetermination symbols and concomitantly introducing the greatest number of alphabet symbols . Processed accordingly, nucleic acid and protein sequences lead to key-words encompassing the salient positions of homologous chains, which can be used for aligning or classifying them, as well as for finding related sequences in data banks. Virology, 1985 Apr 30, 142(2), 223 - 40 Molecular cloning of the Mason-Pfizer monkey virus genome: characterization and cloning of subgenomic fragments; Barker CS et al.; The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described . An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species . The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length . A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus . Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length . These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated . The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses . Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs . Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp . Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA . Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region . No clones containing the pol-coding region have been isolated . In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies . Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected . Sequences more distantly related to the env region were also found in Old World monkeys . Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M-PMV genome, but were only weakly detectable at low stringency. Nucleic Acids Res, 1985 Apr 25, 13(8), 2943 - 58 DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli; Jong AY et al.; In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes . The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E . coli DNA polymerase I or by T4 DNA polymerase . The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors . The template activity could be diminished by treatment with alkali . We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions. Nucleic Acids Res, 1985 Apr 25, 13(8), 2921 - 30 Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells; Chu G et al.; Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1) . This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography . The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt . During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP . This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine . A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X. Nucleic Acids Res, 1985 Apr 25, 13(8), 2789 - 802 A complex of single-strand binding protein and M13 DNA as hybridization probe; Syvanen AC et al.; Single-stranded DNA was complexed to the single-strand binding protein (SSB) of Escherichia coli in a mass ratio of 30:1 . The protein moiety of this complex can be labelled by a number of methods of which we have chosen radio-iodination and biotinylation as examples . The SSB-M13 DNA complexes, labelled to high specific activities, were used as probes in hybridization experiments in which 1.6 X 10(-18) moles of immobilized target DNA were detected . The stability of the hybrids was not severely decreased by the binding of SSB . Analysis of hybrids by electron microscopy showed that complexing of DNA with SSB could be used to allow its subsequent identification in the hybrids. J Biol Chem, 1985 Apr 25, 260(8), 5073 - 80 DNase VII of human placenta . Mechanism studies; Chen GL et al.; The mechanism of the human placental DNase VII, described previously (Hollis, G . F., and Grossman, L . (1981) J . Biol . Chem . 256, 8074-8079) has been investigated in further detail . The enzyme initiates exonucleolytic hydrolysis from the 3'-end of DNA in a nonprocessive, or distributive, manner, regardless of whether the carbohydrate moiety associated with the 3'-terminal nucleotide contains H or OH at its 2' and 3' positions . DNase VII does not have associated RNase H activity; however, it is capable of removing 3'-terminal ribonucleotides . The enzyme also can hydrolyze DNA containing a terminal nucleotide lacking a purine or pyrimidine as well as termini containing noncomplementary nucleotides . DNase VII activity is product-inhibited by deoxynucleoside 5'-monophosphates . From kinetic studies, the mononucleotide deoxyadenylic acid is a noncompetitive inhibitor with a Ki = 0.3 mM . The resemblance of DNase VII to the 3'----5' exonuclease activity of Escherichia coli DNA polymerase I and its possible role in excision repair and proofreading are discussed. J Biol Chem, 1985 Apr 25, 260(8), 4718 - 23 Deletion mapping the yeast TRP5 control region; Moye WS et al.; The yeast gene TRP5 is regulated by the general control system of amino acid biosynthesis . A TRP5-lacZ translational fusion was constructed in order to facilitate assay for TRP5 promoter function and regulation . The chromosomally integrated fusion was derepressed 4-5-fold by lysine limitation . Thus, the TRP5-lacZ fusion is regulated by general control . Deletions were constructed in vitro in the 5'-flanking region of cloned TRP5-lacZ . These deletions localized the promoter region to within 188 base pairs of the transcription start site . Two separable subregions of the promoter were localized by further deletion mapping . The boundaries of the promoter regions are approximately -188 to -114 and -91 to -29 . The TATA box is contained within the downstream promoter region . Regulatory regions essential for general control overlap with these promoter elements . Each of two separable regulatory regions contains a copy of the TGACT repeat (Donahue, T . F., Daves, R . S., Lucchini, G., and Fink, G . R . (1983) Cell 32, 89-98) previously implicated in regulation of HIS4 by general control. J Biol Chem, 1985 Apr 25, 260(8), 4984 - 94 Mechanism of strand passage by Escherichia coli topoisomerase I . The role of the required nick in catenation and knotting of duplex DNA; Dean FB et al.; We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings . The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold . The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick . DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently . The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements . Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick . Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break . For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation . We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment. J Biol Chem, 1985 Apr 25, 260(8), 4975 - 83 Duplex DNA knots produced by Escherichia coli topoisomerase I . Structure and requirements for formation; Dean FB et al.; We investigated systematically the knotting of nicked circular duplex DNA by Escherichia coli topoisomerase I . Agarose gel electrophoresis of knots forms a ladder of DNA bands . Each rung is made up of a variety of knots with the same number of nodes, or segment crossings; knots in adjacent rungs differ by one node . We extended the technique of electron microscopy of recA protein-coated DNA to the visualization of the complex knots tied by topoisomerase I . The striking result is that the enzyme produces every knot theoretically possible . The requirement for excess enzyme to form complex knots suggests a role for topoisomerase I in contorting the DNA in addition to promoting strand passage . We conclude that nodes formed are equally likely to be positive or negative and that topoisomerase I can pass DNA strands through a transient enzyme-generated break without regard to orientation of the passing strand . The results are interpreted in terms of a formulation for the topological requirements for knotting. J Biol Chem, 1985 Apr 25, 260(8), 4901 - 7 The defective proton-ATPase of uncD mutants of Escherichia coli . Two mutations which affect the catalytic mechanism; Duncan TM et al.; The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis . Both mutant enzymes showed reduced affinity for ATP at the first catalytic site . While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release . Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes . Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal . In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent . The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis . Taken together with previous studies of uncA mutant F1-ATPases (Wise, J . G., Latchney, L . R., Ferguson, A . M., and Senior, A . E . (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits. J Biol Chem, 1985 Apr 25, 260(8), 4807 - 14 H+-ATPase of Escherichia coli . An uncE mutation impairing coupling between F1 and Fo but not Fo-mediated H+ translocation; Mosher ME et al.; The uncE114 mutation from Escherichia coli strain KI1 (Nieuwenhuis, F . J . R . M., Kanner, B . I., Gutnick, D . L., Postma, P . W., and Van Dam, K . (1973) Biochim . Biophys . Acta 325, 62-71) was characterized after transfer to a new genetic background . A defective H+-ATPase complex is formed in strains carrying the mutation . Based upon the genetic complementation pattern of other unc mutants by a lambda uncE114 transducing phage, and complementation of uncE114 recipients by an uncE+ plasmid (pCP35), the mutation was concluded to lie in the uncE gene . The uncE gene codes for the omega subunit ("dicyclohexylcarbodiimide binding protein") of the H+-ATPase complex . The mutation was defined by sequencing the mutant gene . The G----C transversion found results in a substitution of Glu for Gln at position 42 of the omega subunit in the Fo sector of the H+-ATPase . The substitution did not significantly impair H+ translocation by Fo or affect inhibition of H+ translocation by dicyclohexylcarbodiimide . Wild-type F1 was bound by uncE114 Fo with near normal affinity, but the functional coupling between F1 and Fo was disrupted . The uncoupling was indicated by an H+-leaky membrane, even when saturating levels of wild-type F1 were bound . Disassociation of F1 from Fo under conditions of assay did partially contribute to the H+ leakiness, but the major contributor to the high H+ conductance was Fo with bound F1 . The F1 bound to uncE114 membranes exhibited normal ATPase activity, but ATP hydrolysis was uncoupled from H+ translocation and was resistant to inhibition by dicyclohexylcarbodiimide . The F1 isolated from the uncE114 mutant was modified with partial loss of coupling function . However, this modification did not account for the uncoupled properties of the mutant Fo described above, since these properties were retained after reconstitution of mutant membrane (Fo) with wild-type F1. Biochemistry, 1985 Apr 23, 24(9), 2268 - 74 On the structural specificity of puromycin binding to Escherichia coli ribosomes; Weitzmann C et al.; We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified {Nicholson, A . W., Hall, C . C., Strycharz, W . A., & Cooperman, B . S . (1982) Biochemistry 19, 3809-3817, and references cited therein} by examining the interactions of a series of adenine-containing compounds with these sites . We have used as measures of such interactions the inhibition of {3H}puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity . For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation {Kerlavage, A . R., Weitzmann, C., Hasan, T., & Cooperman, B.S . (1983) J . Chromatogr . 266, 225-237} . We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7 . Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site . Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides . They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety. Biochemistry, 1985 Apr 23, 24(9), 2284 - 91 Alternative conformers of 5S ribosomal RNA and their biological relevance; Christensen A et al.; Different conformational states of Escherichia coli 5S ribosomal RNA that may participate in protein biosynthesis have been either detected experimentally or predicted on the basis of phylogenetic sequence comparisons . The A conformer exists in a high-salt form (AH) that binds ribosomal proteins and assembles into the 50S subunit and in a low-salt form (AL), of uncertain biological relevance, that binds at least one ribosomal protein and differs in tertiary structure from the AH form . Experimentally, the AH form has been investigated comprehensively and the AL form partially . There is also a B conformer that exhibits an altered secondary structure and does not assemble with ribosomal proteins . For this conformer exhibits an altered secondary structure and does not assemble with ribosomal proteins . For this conformer to be functionally active, it must be both discrete and universal among 5S RNAs . Here, we examine its structure by employing single and double strand specific ribonucleases and nucleotide-specific chemical reagents . We demonstrate that the B form exhibits a secondary structure only a part of which is both universal and conformationally homogeneous, and we conclude, therefore, that the whole B form cannot participate in protein biosynthesis . We note, however, that progressive structural changes occur during the transitions AH----AL----B and provide evidence that the structural alteration during the transition AH----AL may be universal, which reinforces the view that the AL form is of biological relevance. Biochemistry, 1985 Apr 23, 24(9), 2245 - 53 Studies on transcription of 3'-extended templates by mammalian RNA polymerase II . Parameters that affect the initiation and elongation reactions; Dedrick RL et al.; Addition of short sequences of dCMP residues to the 3'-OH end of duplex linear DNAs allows rapid and efficient transcription to be initiated at these sites by purified mammalian RNA polymerase II {Kadesch, T . R., & Chamberlin, M . J . (1982) J . Biol . Chem . 257, 5286-5295} . The use of such tailed DNA templates should allow biochemical studies on transcription elongation and termination with almost any desired DNA sequence . However, in vitro transcription with RNA polymerase II is aberrant in that the DNA template is not re-formed after transcription; rather, the DNA strands are separated, and most of the RNA product is found as a DNA-RNA hybrid . To better understand the factors that affect the process of transcription with these tailed DNA templates, we have varied a number of parameters that might be expected to play a role in the reaction . RNA polymerase II preparations from calf thymus, HeLa cells, and Drosophila all fail to displace the product RNA . However, RNA polymerase II from wheat germ gives only free RNA as a product, as does the Escherichia coli RNA polymerase . Hence, the displacement of the nascent RNA from a transcription complex seems to depend on some intrinsic property of the polymerase itself and not simply on the nature of the template . Variation of reaction conditions, or of the divalent metal ion, does not restore the renaturability of the DNA template . However, variation of the duplex 3'-terminal sequence of the template led to significant alterations . In general, GC-rich sites enhanced the displacement of the nascent RNA, while AT-rich sites enhanced formation of the DNA-RNA hybrid.(ABSTRACT TRUNCATED AT 250 WORDS) Thromb Haemost, 1985 Apr 22, 53(2), 252 - 4 Influence of cyproheptadine on endotoxin-induced disseminated intravascular coagulation (DIC) in weaned pigs; Nowak G et al.; Changes in the coagulation system typical of consumption reaction, microthrombosis in the lungs and other organs and haemodynamic disturbances caused by infusion of endotoxin in weaned pigs were prevented by treatment with the serotonin receptor antagonist cyproheptadine. FEBS Lett, 1985 Apr 22, 183(2), 275 - 8 3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis; Chidgeavadze ZG et al.; It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase . The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase alpha . The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs . dNTP(3'F) produce a more clear sequence pattern than do ddNTPs. J Mol Biol, 1985 Apr 20, 182(4), 597 - 606 Kinetics and importance of the dimerization step in the folding pathway of the beta 2 subunit of Escherichia coli tryptophan synthase; Blond S et al.; During its folding, the polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (L-serine hydrolyase (adding indole) EC 4.2.1.20) undergoes dimerization . To decide whether this dimerization precedes or follows the formation of the native, functional, tertiary structure of the polypeptide chain, the kinetics of renaturation of beta 2 are studied by monitoring both the regain of native conformation and the dimerization . Dimer formation is followed by the increase of the fluorescence polarization, or by energy transfer between a fluorescence donor and a fluorescence acceptor, which occur upon association of adequately labelled beta chains . Renaturation is followed by the regain of functional properties of beta 2, i.e . its ability to bind pyridoxal-5'-phosphate or to form a fluorescent ternary complex with this coenzyme and L-serine . It is shown that for beta 2 the dimerization obeys first-order kinetics, presumably because it occurs rapidly after a rate-limiting isomerization of the monomer . The dimerization is followed by another isomerization, taking place within the dimer, which leads to the formation of the pyridoxal-5'-phosphate binding site . Still another, slow, isomerization reaction involving the F1 (N-terminal) domain completes the renaturation . With a modified form of beta 2 (trypsin-nicked beta 2) where this isomerization of F1 can be made to occur before the dimerization, the dimer is also shown to appear before the functional properties . It is concluded that a non-native dimer indeed exists as an obligatory intermediate on the folding pathway of nicked beta 2 and of beta 2, and that interdomain interactions are needed to force the polypeptide chains into their native conformations. J Mol Biol, 1985 Apr 20, 182(4), 529 - 40 Sense codons are found in specific contexts; Yarus M et al.; The sequence environment of codons in structural genes has been investigated statistically, using computer methods . A set of Escherichia coli genes with abundant products was compared with a set having low gene product levels, in order to detect potential differences associated with expression . The results show striking non-randomness in the nucleotides occurring near codons . These effects are, unexpectedly, very much larger and more homogeneous among the genes with rare products . The intensity of effects in weakly expressed genes suggests that such non-random sequence environments decrease expression . In the weakly expressed set of genes, the 5' neighbor of a codon, and all positions of the 3' neighbor codon are biased . In the highly expressed genes, the first nucleotide of the next codon is a uniquely affected site . The distribution of non-randomness in weakly expressed genes suggests that sequence bias is primarily due to a constraint acting directly on the secondary or tertiary structure of the codon/anticodon . In highly expressed genes, the observed bias suggests an interaction between the codon/anticodon and a site outside the codon/anticodon . Much of the tendency to non-random near-neighbor sequences in weakly expressed genes can be ascribed to a correlation between nearby nucleotides and the wobble nucleotide of the codon, despite the fact that selection of such correlations will alter the amino acid sequence . The favored pattern, in genes expressed at low level, is R YYR or Y RRY . R indicates purine, Y indicates pyrimidine; the space is the boundary between codons . It seems likely that this preference for nearby sequences is the physical basis of the genetic context effect . Under this assumption such sequence biases will affect expression . On this basis, we predict new sites for contextual mutations which decrease expression, and suggest strategy for the design of messages having optimal translational activity. J Mol Biol, 1985 Apr 20, 182(4), 611 - 2 Apparent alteration in properties of arl mutants of Escherichia coli; Hays JB et al.; The published properties of lambda phages grown in Escherichia coli arl mutants, and plasmids maintained in them, included increased homologous recombination, decreased DNA-cytosine methylation, and increased sensitivity of DNA to nuclease S1 . Some of these properties now appear altered; others remain approximately as published. J Mol Biol, 1985 Apr 20, 182(4), 495 - 508 Autogenous regulation of transcription termination factor Rho; Barik S et al.; We present evidence that the transcription termination factor Rho is autogenously regulated in Escherichia coli . The steady-state level of Rho is increased approximately tenfold in rho mutant cells . In the rho+ revertants, the content of Rho is similar to the wild-type level . A rho-/rho+ merodiploid produces equimolar amounts of the mutant and the wild-type Rho polypeptides, both at a reduced level compared to the mutant . The steady-state level of rho messenger RNA is also increased in a rho mutant . A rho-galK transcriptional fusion produces at least tenfold more galactokinase in a rho- strain than in a rho+ strain . In vitro, in a coupled transcription-translation system, the synthesis of Rho protein is specifically inhibited by wild-type Rho but not by Rho15 mutant protein . Anti-Rho antibody specifically stimulates Rho synthesis in the rho+ extract but not in a rho- extract . We suggest that the autogenous regulation of Rho involves premature transcription termination within the rho gene . Regulation of Rho level may provide the cell a mechanism to modulate the expression of genes which are separated from their promoters by Rho-dependent termination signals. Biochim Biophys Acta, 1985 Apr 19, 824(4), 304 - 12 Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum; Guyer RB et al.; Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75 . These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively . Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+ . These activities are apparently specific only for AP sites in DNA . The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs . Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I . Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars . It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation . Many of the properties of these D . discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells . They will be of use in the characterization of excision repair in this organism. Biochem Biophys Res Commun, 1985 Apr 16, 128(1), 257 - 64 Comparison of the biological properties of purified natural and recombinant human interleukin-2; Naruo K et al.; We compared the biological properties of the purified recombinant human IL-2 derived from E . coli with those of purified natural IL-2 . Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells . No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography. Biochem Biophys Res Commun, 1985 Apr 16, 128(1), 155 - 62 The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property; Aris JP et al.; We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane . Minicells containing {35S}-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate . ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits . Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits . About half of this amount (19%) of beta is non-specifically bound to the membrane . Interaction of beta with the membrane is not mediated by the amino terminal portion of beta. Biochem Biophys Res Commun, 1985 Apr 16, 128(1), 171 - 8 Study of antigenic epitopes recognized by monoclonal antibodies to recombinant interferon-gamma; Liang CM et al.; Seven hybridomas (BG 1-7) which secreted monoclonal antibodies against recombinant interferon-gamma were produced . The ascites fluids containing four of the seven monoclonal antibodies (BG 1-4) neutralized the antiviral activity of both natural and recombinant interferon-gamma . Competition between labeled and unlabeled monoclonal antibodies for interferon-gamma in a solid phase immunoassay showed that BG 1 was competed by both BG 3 and BG 4 but not by BG 2; BG 2 was competed by BG 3 but not by BG 1 nor by BG 4 . These results suggest that human interferon-gamma has at least two antigenic epitopes; one of the epitopes reacted with BG 1 & BG 4 while the other reacted with BG 2; BG 3 either binds to a region overlapping with the other two epitopes or reacts with both epitopes . The antigenic epitopes recognized by these four neutralizing monoclonal antibodies are likely at or closely related to the active sites of interferon-gamma. Eur J Biochem, 1985 Apr 15, 148(2), 271 - 5 Does UGA suppressor tRNATrp from Escherichia coli have a unique CCA anticodon sequence? Delamarche C, Buckingham RH. The properties of the UGA-suppressor tRNATrp (anticodon CCA) from Escherichia coli has greatly influenced ideas about the specificity of codon-anticodon interactions showing that the anticodon sequence is not the sole determinant . However, a recent hypothesis for the mechanism of suppression by this tRNA proposes that the base change in position 24, in the dihydrouridine stem, leads to a change in translational specificity of the tRNA by increasing post-transcriptional modification of cytidine 34, in the anticodon wobble position {M . Yarus (1982) Science (Wash . DC) 218, 646-652} . The enzyme postulated to do this normally modifies C34 of a minor isoleucine isoacceptor specific for AUA codons . This modification should reduce reading of G in the third codon position: affinity chromatography on columns containing immobilised tRNAPro (anticodon VGG) has therefore been employed to isolate an enriched population of the putative suppressor species, if such a sub-population exists . The results obtained are difficult to reconcile with the presence of a subfraction, modified post-transcriptionally in C34, responsible for the suppression of UGA . This argues in favour of the previously advanced hypothesis for the mechanism of suppression, which depends on events outside the codon-anticodon interaction itself. Nucleic Acids Res, 1985 Apr 11, 13(7), 2583 - 601 Novel non-suppressing mutants of Escherichia coli tRNATyr su+3; Furdon P et al.; Several addition and deletion mutations were constructed in the region of the gene for Escherichia coli tRNATyr su+3 corresponding to the dihydrouracil loop of the mature tRNA . None of these resulting mutants had detectable suppressor function compared to the parent gene yet some directed the synthesis of mature tRNA . These latter mutants may affect the ability of the tRNA to be aminoacylated or to interact with the translational machinery on the ribosome. Nucleic Acids Res, 1985 Apr 11, 13(7), 2519 - 31 Analysis of the promoters for the two immunity genes present in the ColE3-CA38 plasmid using two new promoter probe vectors; Chak KF et al.; We have constructed two new promoter probe vectors which carry a polylinker derived from plasmid pUC19 proximal to the 5' end of a promoter-less galactokinase gene . Using these two vectors we have demonstrated that the ColE3imm gene and the ColE8imm gene present on the ColE3-CA38 plasmid have their own promoters, independent of the SOS promoter of the colicin E3 structural gene . The activity of two terminators, one located proximal to the 5' end of the ColE8imm gene, the other located proximal to the 5' end of the lys gene, were shown by a comparison of the galactokinase activity conferred by several of the recombinant plasmids. Nucleic Acids Res, 1985 Apr 11, 13(7), 2457 - 68 Deletion analysis of the Escherichia coli lactose promoter P2; Yu XM et al.; The Escherichia coli lactose (lac) operon transcription control region includes at least two sequences which are recognized by RNA polymerase holoenzyme in vitro, the normal lac promoter (termed P1) and an overlapping upstream promoter (termed P2) . The structure of the P2 and the effect of RNA polymerase interaction at P2 on the association of RNA polymerase with P1 was analyzed by the isolation and characterization of various mutations at P2 . A set of deletions with varying lengths of DNA between the lac P2 -10 region and a "-35 region" contributed by the vector DNA were constructed . In vitro studies indicate that as the spacing between the -10 region and "-35 region" is increased from 16 to 22 base pairs (bp), the steady state occupancy as measured by exonuclease III protection experiments and the ability to initiate transcripts from P2 decrease . Studies were also conducted using a single base pair insertion and a two base pair deletion between the natural -35 and -10 regions of P2 . The mutation which decreases the in vitro occupancy and transcription initiation potential of P2 does not significantly affect the steady state in vitro occupancy of P1 nor the in vivo expression of the lac operon . These results are not consistent with the model that RNA polymerase occupancy at P2 competes with the P1 expression and therefore that this competition plays a role in cAMP bound catabolite gene activator protein (CAP-cAMP) control of the lac operon. Nucleic Acids Res, 1985 Apr 11, 13(7), 2337 - 56 Primary sequence and partial secondary structure of the 12S kinetoplast (mitochondrial) ribosomal RNA from Leishmania tarentolae: conservation of peptidyl-transferase structural elements; de la Cruz VF et al.; The sequence of the 1173 nt 12S kinetoplast ribosomal RNA from Leishmania tarentolae was determined from the maxicircle DNA sequence, and the 5' and 3' ends localized by primer runoff and S1 nuclease protection experiments . The gene was shown to be free of introns by S1 nuclease analysis . A partial secondary structure model of the 12S RNA molecule is presented which is equivalent in certain respects to the corresponding portions of the Escherichia coli 23S ribosomal RNA model . Domain II of the E . coli model is completely missing in the kinetoplast model with the exception of several phylogenetically conserved stems and one loop . There is a striking conservation of the functionally important peptidyl-transferase region except for the deletion of a few stems and loops . The 12S RNA is the smallest large subunit ribosomal RNA described to date. J Biol Chem, 1985 Apr 10, 260(7), 4122 - 7 Rapid kinetics of Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles . The effect of bilayer curvature on leakage; Morris SJ et al.; We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion . Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation . Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation . Thus fusion takes place in the microsecond time domain . Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse . On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size . Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made . NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals . Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts. J Biol Chem, 1985 Apr 10, 260(7), 4075 - 81 The Escherichia coli adenylate cyclase complex . Stimulation by potassium and phosphate; Liberman E et al.; In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot . Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate . Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells . The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP . While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells . Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction . The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated . Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS . A model for dual regulation by glucose of adenylate cyclase activity is proposed . This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate. J Biol Chem, 1985 Apr 10, 260(7), 3937 - 40 Secondary structure of human leukocyte interferon from Raman spectroscopy; Williams RW; Raman spectra were taken of human alpha (leukocyte) interferon subtype A (HuIFN-alpha A) purified from extracts of transformed Escherichia coli . Quantitative analysis of the conformationally sensitive amide I band indicates that IFN (interferon)-alpha A is 75 +/- 5% helical and 7 +/- 4% beta-strand . An independent analysis of the amide III spectrum indicates 71 +/- 5% helix and 10 +/- 6% beta-strand . These results differ with a recently proposed three-dimensional model based on secondary structure predictions derived from sequence and with circular dichroism measurements . The Raman spectrum of IFN-alpha A is compared with the spectra of several other helical proteins: hemerythrin, intestinal calcium-binding protein, melittin, and insulin. J Biol Chem, 1985 Apr 10, 260(7), 4236 - 42 DNA sequence analysis of the dye gene of Escherichia coli reveals amino acid homology between the dye and OmpR proteins; Drury LS et al.; Mutation of the dye gene of Escherichia coli results in sensitivity of dyes, envelope protein changes, loss of expression of alkaline phosphatase, and reduced transcription of sex factor F genes . We have determined the DNA sequence of a 1.4-kilobase pair fragment encompassing the dye gene . The coding sequence of dye was identified as an open reading frame coding for a protein of Mr 27,346 . A sequence of 54 residues at the amino terminus was extremely acidic, with 12 aspartic plus glutamic acid residues and only 2 lysine plus arginine residues . A sequence of 19 adjacent residues near the center of the protein was identical, except for one mismatch, with a sequence in the OmpR protein, involved in controlling the amounts of the major outer membrane proteins OmpF and OmpC at the level of transcription . 28% of the Dye protein was homologous with OmpR . The positions of dye and ompR on the genetic map were indicative of a gene duplication . It seems likely, therefore, that the Dye and OmpR proteins are related, and Dye may thus be involved in the osmoregulation of envelope protein genes as well as being required for sex factor gene expression . The Dye protein itself, like OmpR, was shown not to be an envelope protein . A second open reading frame on the other DNA strand may use the same transcription termination site as dye. J Biol Chem, 1985 Apr 10, 260(7), 4091 - 7 sn-1,2-Diacylglycerol kinase of Escherichia coli . Purification, reconstitution, and partial amino- and carboxyl-terminal analysis; Loomis CR et al.; The sn-1,2-diacylglycerol kinase structural gene from Escherichia coli was demonstrated to be the dgkA locus previously sequenced (Lightner, V . A., Bell, R . M., and Modrich, P . (1983) J . Biol . Chem . 258, 10856-10861) . The dgkA gene product was shown by maxicell analysis to be an Mr = 14,000 membrane-bound protein . When dgkA was placed on a hybrid plasmid under control of the lambda pL promoter, a 100-fold overproduction of diacylglycerol kinase activity was obtained . Diacylglycerol kinase was solubilized from membranes with 2-propanol/heptane/trifluoroacetic acid and purified to near homogeneity by high performance liquid chromatography . Activity was reconstituted in a mixed micellar assay containing beta-octylglucoside, cardiolipin, and sn-1,2-dioleoylglycerol . Amino acid analysis, partial NH2-terminal analysis and COOH-terminal analysis permitted alignment of the polypeptide on the sequenced gene . The data establish that dgkA is the structural gene for the diacylglycerol kinase and establish the primary structure of the enzyme of 122 residues, 13,245 daltons . Secondary structure analysis predicted a protein conformation consisting of three transmembrane alpha-helical segments, an amphipathic helix, and an alpha-helix . Taken together, the predicted helical segments comprise more than 75% of the polypeptide. J Biol Chem, 1985 Apr 10, 260(7), 3906 - 9 Viral Harvey ras p21 expressed in Escherichia coli purifies as a binary one-to-one complex with GDP; Poe M et al.; The ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation . Viral Harvey ras p21, cloned into Escherichia coli HB101 lambda/pRAS1, has been purified to homogeneity by a three-step procedure . The purified E . coli p21 can be bound by a monoclonal antibody to viral Harvey ras p21 and has an amino acid composition consistent with that predicted from its DNA sequence . Purified E . coli p21 has been shown by HPLC analysis on an ion-exchange column to contain near stoichiometric amounts of GDP . This noncovalently associated GDP is seen in the UV absorption spectrum of the purified protein . The noncovalently associated GDP acts as a competitive inhibitor of the interaction of added guanine nucleotides with p21. J Biol Chem, 1985 Apr 10, 260(7), 4378 - 83 Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein; Li XM et al.; Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated . Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP . Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core . The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP . Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase . Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment . One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex . Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used. Biochemistry, 1985 Apr 9, 24(8), 2072 - 6 Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli; Brody RS et al.; Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 {Prasher, D . C., Conarro, L., & Kushner, S . R . (1983) J . Biol . Chem . 258, 6340-6343} . The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus . This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate . This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined. Biochemistry, 1985 Apr 9, 24(8), 1856 - 61 Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay; Jorns MS et al.; Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates {oligo(dT)n} containing 4-18 thymine residues . This rate is identical with that observed with polythymidylate and with native DNA . The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues . A decrease in affinity is detectable with oligo(dT)n when n = 4-6 . The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues . Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9 . Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends . The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers . The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 {p(dT)2} since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end . That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer . Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired. Biochemistry, 1985 Apr 9, 24(8), 1849 - 55 Binding of Escherichia coli DNA photolyase to UV-irradiated DNA; Sancar GB et al.; Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation . In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate . Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay . We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded . The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5 . Although photolyase is retained by the nitrocellulose filters with near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34 . The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter. FEBS Lett, 1985 Apr 8, 183(1), 52 - 4 Degradation of somatomedins by the thioredoxin system; Enberg G et al.; The insulin disulfide reducing thioredoxin system from E . coli was used to investigate a possible mechanism of degradation for the two somatomedins, insulin-like growth factor I and II (IGF-I and -II) . The amounts of IGF-I and -II remaining after degradation were measured by use of human placenta radioreceptor assay . The results show that both IGF-I and -II were as sensitive to disulfide reduction as insulin. FEBS Lett, 1985 Apr 8, 183(1), 99 - 102 Identification of the stable free radical tyrosine residue in ribonucleotide reductase . A sequence comparison; Sjoberg BM et al.; The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center . An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important . Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved . We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center. J Theor Biol, 1985 Apr 7, 113(3), 425 - 39 Maximum likelihood estimation of subsequence conservation; Lawrence CE et al.; A statistical method is presented for comparing protein sequences by partitioning the polymers and estimating each subsegment's degree of conservation . Conservation is measured as a function of the number of transitions occurring in the underlying time homogeneous Markov process assumed to govern amino acid mutations . The Markovian assumption also permits estimation of the ancestral sequence . Partitioning and estimation are carried out via maximum likelihood . The method is contrasted with the commonly utilized percent homology measure . A moving likelihood ratio plot to aid in identifying regions of high conservation is suggested as an analogue to moving hydrophobicity plots . An application is presented which identifies highly conserved regions in thymidylate synthase from L . casei and E . coli. J Mol Biol, 1985 Apr 5, 182(3), 467 - 8 Preliminary X-ray diffraction studies of acyl carrier protein from Escherichia coli; McRee DE et al.; Crystals of the acyl carrier protein of Escherichia coli have been grown and analyzed by X-ray diffraction . The crystals grow in space group C2 with unit cell dimensions a = 46.8 A, b = 52.1 A, c = 47.3 A and beta = 93.2 degrees . An isomorphous derivative, HgCl2, has been identified and characterized. J Mol Biol, 1985 Apr 5, 182(3), 411 - 7 Point mutations that affect translation initiation in the Escherichia coli gal E gene; Dreyfus M et al.; This paper describes the selection and characterization of several mutations in the Escherichia coli galactose operon that affect translation initiation of the galE gene but are located outside of the Shine-Dalgarno sequence and the initiator codon . One mutation lies in the gal promoter region and shifts transcription initiation from the galP1 to the galP2 promoter . This results in a gal messenger that is five nucleotides longer and that is translated threefold more efficiently in vivo . This accords with previous observations from in vitro experiments which showed that the longer gal messenger was better translated (Queen & Rosenberg, 1981) . The other mutations that affect galE translation are located in the coding sequence immediately downstream from the initiator codon . In contrast to the promoter mutation, these cause alterations in galE expression only when the gene carries a mutated initiator codon or Shine-Dalgarno sequence and have no effect on the wild-type galE gene . These findings are discussed with respect to our present knowledge of translation initiation mechanisms. Science, 1985 Apr 5, 228(4695), 93 - 6 Expression in Escherichia coli of open reading frame gene segments of HTLV-III; Chang NT et al.; Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods . In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients . Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100 . The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus . Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins . Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified . DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome. J Mol Biol, 1985 Apr 5, 182(3), 397 - 409 Analysis of the requirements for transcription pausing in the tryptophan operon; Fisher RF et al.; RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide . Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon . We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro . A model is presented that accounts for many of the findings obtained in this and other pausing studies. Nature, 1985 Apr 4-10, 314(6010), 452 - 4 Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences; Takeda S et al.; The specificity of monoclonal antibodies provides a powerful diagnostic and therapeutic tool in investigating human neoplasia . Radiological scanning and immunotherapy with mouse tumour-specific monoclonal antibodies have been applied to patients with some success, but a major problem is the neutralization of the mouse antibody induced by repeated administration of heterologous antibodies . To avoid or reduce such immune reactions, chimaeric immunoglobulins consisting of mouse variable (V) and human constant (C) regions can be synthesized . We have constructed a recombinant retrovirus DNA carrying genomic heavy-chain (H) variable-diversity joining (VH-D-JH) and C gamma 1 genes from different species and show here that the chimaeric intervening sequences are spliced out precisely . This procedure provides a useful method to construct the chimaeric mouse-human immunoglobulin gene to be expressed in Escherichia coli, yeast and animal cells . Unexpectedly, a hidden splice donor site in the 5'-flanking region of a human VH gene is used in place of the donor site of the leader sequence exon, resulting in the formation of the V region without the leader sequence. J Antimicrob Chemother, 1985 Apr, 15(4), 489 - 94 The monitoring of serum chloramphenicol levels in children with severe infections; Ekblad H et al.; Serum chloramphenicol levels were evaluated in 52 children with severe infection treated intravenously with chloramphenicol succinate and orally with chloramphenicol palmitate, chloramphenicol monostearoylglycolate or chloramphenicol in capsules . Effective serum levels were recorded with all chloramphenicol preparations . The variability was largest with chloramphenicol monostearoylglycolate . In a case of neonatal Escherichia coli meningitis good serum levels of chloramphenicol were achieved with chloramphenicol palmitate orally, supporting the view that oral chloramphenicol palmitate can be used to treat serious infections in this age group . Our data and those in the literature show that monitoring of serum chloramphenicol levels in neonates is necessary . After the neonatal period monitoring of serum chloramphenicol levels is useful in avoiding too high concentrations . On the other hand, toxic effects of high concentrations can be recognized from reticulocyte and haemoglobin, neutrophil and platelet counts, which should be performed every three to four days. J Antibiot (Tokyo), 1985 Apr, 38(4), 505 - 12 Cloning of mitochondrially determined oligomycin resistance, oli-1 in yeast; Mabuchi T et al.; An Escherichia coli-yeast shuttle vector pOL 221 is described, which consists of pBR 322 and a yeast mitochondrial fragment . The mitochondrial insert in pOL 221 possessed a mitochondrial autonomously replicating sequence (ARS) and a single Sst II site . The ARS function of pOL 221 was demonstrated by insertion of LEU2 gene into pOL 221 (giving pOL 2211) followed by the transformation of leu- yeast cells . By use of this vector, chimeric plasmids pOL 37 and pOL 379 (a derivative of pOL 37 inserted with LEU2 gene), carrying the yeast mitochondrial oli-I gene were constructed . Marked instability was observed on the maintenance of pOL 37 in E . coli under non-selective conditions . pOL 37 can, however, be maintained in E . coli in the presence of tetracycline . pOL 2211, pOL 379 and other pOL plasmids showed mitotic instability in yeast . Transformants of oligomycin sensitive yeast cells with pOL 37 and pOL 379 did not show oligomycin resistance . The absence of recombination between the cloned mitochondrial gene and the host mitochondrial gene is discussed. Cardiovasc Res, 1985 Apr, 19(4), 201 - 5 Hepatic trapping of red cells in canine endotoxin shock: a variable phenomenon after splenectomy; Teule GJ et al.; We studied in 20 splenectomised dogs the incidence of early intravascular hepatic pooling after administration of E . coli endotoxin . Autologous red cells were labelled in vitro with 99mTc . Bloodpool imaging and haemodynamic measurements were performed simultaneously . Changes in hepatic red cell volume were estimated from alterations in hepatic activity . In 10 dogs, hepatic red cell activity increased considerably (156 to 359% of the basal value) . In the remaining animals the hepatic activity did not change markedly or even decreased . The decline in arterial pressure and cardiac output seemed more pronounced in dogs with clear evidence of hepatic pooling . However no significant differences in absolute haemodynamic values could be demonstrated between dogs with and without pooling . It is concluded that an hepatic outflow block is not a constant feature of canine endotoxin shock . Absolute haemodynamic values do not depend on the presence of an outflow block . Thus the presence of hepatic pooling need not make the canine model inappropriate for studies on human sepsis. Nippon Geka Gakkai Zasshi, 1985 Apr, 86(4), 400 - 3 {Alanine metabolism in the skeletal muscle and plasma in endotoxemia}; Ishida K et al.; Utilizing a 24 hour fasting rabbit (N = 30), we measured free amino acids in the femoral artery and vein and the quadriceps femoris muscle . The endotoxin E . coli 026, 3.0mg/kg (LD100) was injected and free amino acid plasma levels were monitored for 6 hours . Changes in free amino acid plasma levels were variable and marked after endotoxin injection . By 360 min . after endotoxin injection: (a) the rate of increase in free amino acid levels in the femoral artery was 366 mumole/l of alanine, 162 mumole/l of glycine and 85 mumole/l of proline; (b) the rate of increase in free amino acid of the quadriceps femoris muscle was 1376 nmole/g of alanine, 156 nmole/g of glycine and 109 nmole/g of serine; and (c) the femoral arteriovenous difference was -225 nmole/l of alanine, -118 nmole/l of glycine and -77nmole/l of proline . Within 10 min . after endotoxin injection, alanine concentration was higher in the femoral vein . This change in concentration became significant by 60 min . The results show the following: Skeletal muscle appears to be an important source of amino acids for amino acid metabolism during endotoxemia, especially plasma alanine which is closely connected with alanine levels in skeletal muscle. Infect Immun, 1985 Apr, 48(1), 219 - 27 Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs; Takada H et al.; Synthetic lipid A analogs (compounds 404 through 406) were examined for their immunopharmacological activities . These compounds had two amide-bound and two ester-bound (R)-3-hydroxytetradecanoyl groups at the C-2 and C-2' and the C-3 and C-3' positions, respectively, of beta (1-3)glucosamine disaccharide . In all of the in vitro assays, these synthetic compounds exhibited high activities comparable to those of a reference lipid A prepared from Escherichia coli O8:K27 Re-mutant strain F515 . The compounds activated the clotting enzyme cascade of the horseshoe crab, activated the human complement via the classical pathway, caused polyclonal B-cell activation, stimulated the phagocytosis of sheep erythrocytes by murine peritoneal macrophages, and enhanced the migration of human polymorphonuclear leukocytes . They also increased the thymidine uptake of splenocytes of BALB/c nu/nu and C3H/HeN mice but not those of C3H/HeJ (a nonresponder to lipopolysaccharide) . A dephosphorylated derivative, compound 403, was barely active in all of the above assays except for the enhancement of polymorphonuclear leukocyte migration . However, compounds 404 through 406 were feeble in pyrogenicity and could not prepare the local Shwartzman reaction, although they were very lethal to galactosamine-loaded mice . Therefore, synthetic lipid A analogs described here were fully immunopharmacologically active in in vitro assays, but all of them were far less active than natural E . coli F515 lipid A regarding the biological activities characteristic of endotoxic lipopolysaccharides and lipid A's . The high lethal toxicity of compound 406 (1,4'-bisphosphate) to the galactosamine-loaded mice may not reflect its real toxicity to normal mice . In all activities examined, compound 406 was quite comparable to a biosynthetic lipid A precursor, a natural counterpart of compound 406 . The immunopharmacological activities of these newly synthesized lipid A analogs, especially compound 406, were much stronger than those of compounds that had been synthesized earlier by using the originally proposed model of the lipid A structure . The findings described in this report justify the acylation pattern of a disaccharide backbone of lipid A, revised on the basis of recent analytical studies . The low in vivo endotoxic activities of the present lipid A analogs are most probably due to the fact that the kinds of acyl groups were different from those of the complete lipid A from E . coli, although there were no differences in the acylation positions on the disaccharide backbone. Cancer Res, 1985 Apr, 45(4), 1753 - 61 Antineoplastic effects in rats of 5-fluorocytosine in combination with cytosine deaminase capsules; Nishiyama T et al.; 5-Fluorocytosine (5-FC) lacks antineoplastic activity in human subjects because of the absence of cytosine deaminase (CDase) in mammalian cells . Intratumoral conversion of 5-FC into 5-fluorouracil (5-FUra) by locally implanted capsules containing CDase followed by systemic administration of 5-FC can be expected to induce antineoplastic activity at a local site with minimal systemic toxicity . In vitro and in vivo experiments were performed to evaluate this hypothesis . Spectrophotometric analysis confirmed the deamination of 5-FC to 5-FUra by CDase extracted from cultivated Escherichia coli . In vitro studies showed that 5-FC combined with CDase induced significant growth-inhibitory effects on the cultured glioma cells . An active CDase capsule, made of cellulose tubing, was newly designed for local implantation . 5-FC concentrations in the s.c . tumors of the rats given these CDase capsules, followed by 5-FC administration, showed a sufficient amount of delivery of 5-FC to the tumor tissue . 5-FUra appearing in the tumor reached the level of 8.0 micrograms/g at 2 h and stayed at more than 1.0 microgram/g at between 1 and 6 h . Significant reduction of the tumor growth and cytotoxic changes were observed . The passive cutaneous anaphylaxis reaction demonstrated no allergic reaction to the host due to the capsule . These results suggest that this chemotherapeutic method is effective for human brain tumors. J Biochem (Tokyo), 1985 Apr, 97(4), 1247 - 50 Use of a phoB'-'lacZ fusion gene to determine the N-terminal amino acid sequence of the phoB protein and to prepare antiserum against the protein; Sugita T et al.; phoB is a positive regulatory gene of the phosphate regulon of Escherichia coli . A plasmid carrying a phoB'-'lacZ fusion gene was constructed by in vitro recombination . A PhoB-LacZ hybrid protein was purified from the cells carrying the plasmid by monitoring beta-galactosidase activity . The amino-terminal amino acid sequence of the PhoB protein was determined by utilizing the hybrid protein . Antiserum against the PhoB protein was prepared by immunizing rabbits with the hybrid protein . The serum thus prepared showed specificity for both the PhoB protein and beta-galactosidase. EMBO J, 1985 Apr, 4(4), 1067 - 73 Fused lacZ genes code for di-, tri- and tetra-beta-galactosidase in Escherichia coli; Kuchinke W et al.; Plasmids were constructed which carry two, three or four active lacZ genes of Escherichia coli fused head-to-tail in phase . The products of these oligomeric lacZ genes are shown to be polypeptides with expected subunit mol . wts . of 230 kd (di-beta-galactosidase), 350 kd (tri-beta-galactosidase) and 460 kd (tetra-beta-galactosidase) . Di-beta-galactosidase has the same enzymatic activity as the wild-type enzyme . It subunits are practically not degraded proteolytically in vivo . It aggregates predominantly to a dimer which has the same sedimentation constant as the wild-type tetrameric enzyme . Furthermore, it is more heat stable than the wild-type enzyme . Tri- and tetra-beta-galactosidase have strongly reduced enzymatic activities and are largely degraded . Our experiments lead us to propose that covalent joining of two subunits through proper gene duplication may possibly be an intermediate in the evolution of self aggregation of homo-oligomeric proteins. EMBO J, 1985 Apr, 4(4), 1049 - 52 Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors; Vijgenboom E et al.; Read-through of nonsense codons has been studied in wild-type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF-Tu . The two phenomena differ essentially . Readthrough of UGA in wild-type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid-borne EF-Tu . This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF-Tu . Strains of E . coli harbouring mutant species of the elongation factor EF-Tu suppress the nonsense codons UAG, UAA and UGA . Suppression shows a codon context dependence . It requires the combined action of two different EF-Tu species: EF-TuAR(Ala 375----Thr) and EF-TuBo(Gly 222----Asp) . Cells harbouring EF-TuAR(Ala 375----Thr) and wild-type EF-TuB, or wild-type EF-TuA and EF-TuBo(Gly 222----Asp) do not display suppressor activity . These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors . The requirement for two different mutant EF-Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF-Tu molecules on the ribosome. Can J Physiol Pharmacol, 1985 Apr, 63(4), 315 - 9 Valproic acid disposition in rabbits during chronic treatment with Escherichia coli endotoxin; Thiessen JJ et al.; The disposition of valproic acid (VPA) in rabbits was studied after chronic treatment with Escherichia coli endotoxin . Endotoxin (1-2 micrograms/kg) was administered daily to 10 male New Zealand white rabbits for 5 days . On day 5, 50 mg/kg of VPA was given iv during the time of the peak febrile response . Blood samples were drawn at appropriate time intervals and analyzed for free and total VPA levels as well as plasma proteins and free fatty acids . The data were compared with similar control experiments performed 2 weeks before and after endotoxin treatment . Pharmacokinetic analysis indicated that the changes in free VPA clearance after endotoxin were related to the change in the febrile response during chronic treatment (r = 0.77; p less than 0.05); that is, animals which developed tolerance to the febrile response showed elevated drug clearance, whereas nontolerant animals showed decreased clearance of VPA. Arch Microbiol, 1985 Apr, 141(3), 260 - 5 Tetracycline uptake by susceptible Escherichia coli cells; Argast M et al.; Experiments measuring the initial uptake of commercial (3H) tetracycline exhibit two distinct kinetic phases: a rapid phase followed by a slow phase . (3H) tetracycline purified by chromatography on a Dowex 50WX2 column exhibited only monophasic rapid uptake when tested with susceptible Escherichia coli cells . Cyanide inhibited the uptake of purified (3H) tetracycline only partially while transport of proline and maltose was entirely abolished . Energy independent accumulation of tetracycline may be accounted for by binding to cellular constituents . Uptake of tetracycline--as measured by inhibition of beta-galactosidase synthesis--was strongly affected by a shift in temperature from 37 degrees C to 21 degrees C while carrier-mediated transport systems revealed only minor reductions . Taken together with the non-saturability of tetracycline uptake and the evidence for diffusion of tetracycline through phospholipid bilayers {Argast and Beck (1984) Antimicrob Agents Chemother 26:263-265} these data support the hypothesis that tetracycline enters the cytoplasm by diffusion. J Gen Microbiol, 1985 Apr, 131 ( Pt 4), 935 - 8 Escherichia coli thymine auxotrophs suppressible by carbon dioxide; Dale JW et al.; Two independently-isolated thymine-requiring mutant strains of Escherichia coli were found to possess an unusual phenotype: in the presence of CO2, growth was independent of thymine . The two strains showed different profiles of temperature sensitivity . Glycine, serine and methionine were unable to relieve the thymine-dependence . Both strains were susceptible to trimethoprim under conditions where they were thymine-independent . The results are consistent with the occurrence of partial defects in thymidylate synthase. Eur J Immunol, 1985 Apr, 15(4), 404 - 7 Interferon gamma produced by mitogen-activated T lymphocytes does not directly mediate lymphoproliferation; Wilkinson M et al.; Human interferon gamma (IFN-gamma) endogenously produced during mitogenic stimulation of human peripheral blood mononuclear cells or human T lymphocyte-enriched cultures was neutralized in situ by the addition of a polyclonal antiserum (anti-L) raised in a rabbit against partially purified natural IFN-gamma derived from peripheral blood mononuclear cells . Small decreases in mitogen-induced {3H}thymidine incorporation (lymphoproliferation) were demonstrated under these conditions . However, an antiserum (anti-G) raised in a sheep against highly purified recombinant IFN-gamma (E . coli-derived) which strongly neutralized the antiviral effect of IFN-gamma either had little effect on mitogen-induced lymphoproliferation or caused slight enhancement of mitogenesis . The interleukin 2 responsiveness of activated T lymphocytes following mitogenic stimulation was not found to be different in the presence of anti-G to that of control cultures incubated in the presence of normal sheep serum . These results suggest that IFN-gamma is not a direct requirement for lymphoproliferation. Am J Obstet Gynecol, 1985 Apr 1, 151(7), 967 - 75 Septicemia during pregnancy: a study in different species of experimental animals; Beller FK et al.; When Escherichia coli B6 lipopolysaccharide, 0.2 mg/kg of body weight, was infused into nonpregnant minipigs during a 5-hour period, the animals died after 12 to 16 hours as a result of endotoxic shock . When the same infusion was given to six pregnant minipigs at term, these animals died after only 3 1/2 hours . The decrease in the number of white blood cells, the number of platelets, hematocrit, and clotting factors was not significantly different between the two groups . The acid-base status, however, indicated a much more pronounced metabolic acidosis in the pregnant animals than in the nonpregnant controls . In the pregnant minipigs heart rate, cardiac output, mean arterial pressure, and total peripheral resistance indicated cardiovascular collapse, and the multiple wire, platinum surface electrode revealed a drastic reduction in uterine tissue oxygenation in the pregnant animals . The data support the hypothesis that pregnant animals at term are more susceptible to the harmful effects of lipopolysaccharide . Early death in the pregnant minipigs, however, was not associated with disseminated intravascular coagulation as it is in smaller animals (rat, rabbit, and hamster). J Bacteriol, 1985 Apr, 162(1), 50 - 4 Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B; Bayer MH et al.; The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M) . Phosphoglycerides labeled with {14C}palmitic acid and {3H}serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography . The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane . The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes . This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites . The highest phospholipase C activity was detected in the inner membrane and Int.M fractions . The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains. J Bacteriol, 1985 Apr, 162(1), 286 - 93 Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli; Richarme G; We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation . Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate . Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply . The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone . The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. J Bacteriol, 1985 Apr, 162(1), 196 - 202 Induction of superoxide dismutases in Escherichia coli B by metal chelators; Pugh SY et al.; The effects of metal salts, chelating agents, and paraquat on the superoxide dismutases (SODs) of Escherichia coli B were explored . Mn(II) increased manganese-containing SOD (MnSOD), whereas Fe(II) increased iron-containing SOD (FeSOD) . Chelating agents induced MnSOD but decreased FeSOD and markedly increased the degree of induction seen with Mn(II) . Paraquat also exerted a synergistic effect with Mn(II) . High levels of MnSOD were achieved in the combined presence of Mn(II), chelating agent, and paraquat . All of these effects were dependent on the presence of oxygen . MnSOD, not ordinarily present in anaerobically grown E . coli cells, was present when the cells were grown anaerobically in the presence of chelating agents . These results are accommodated by a scheme which incorporates autogenous repression by the apoSODs and competition between Fe(II) and Mn(II) for the metal-binding sites of the apoSODs . It is further supposed that oxygenation and intracellular O2- production favor MnSOD production because O2- oxidizes Mn(II) to Mn(III), which competes favorably with Fe(II) for the apoSODs. J Biochem (Tokyo), 1985 Apr, 97(4), 993 - 9 Branched-chain amino acid aminotransferase of Escherichia coli: nucleotide sequence of the ilvE gene and the deduced amino acid sequence; Kuramitsu S et al.; The ilvE gene of the Escherichia coli K-12 ilvGEDA operon, which encodes branched-chain amino acid aminotransferase {EC 2.6.1.42}, was cloned . The nucleotide sequence of 1.5 kilobase pairs containing the gene was determined . The coding region of the ilvE gene contained 927 nucleotide residues and could encode 309 amino acid residues . The predicted molecular weight, amino acid composition and the sequence of the N-terminal 15 residues agreed with the enzyme data reported previously (Lee-Peng, F.-C., et al . (1979) J . Bacteriol . 139, 339-345) . From the deduced amino acid sequence, the secondary structure was predicted. J Biochem (Tokyo), 1985 Apr, 97(4), 1259 - 62 Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene; Kuramitsu S et al.; The nucleotide sequence of the aspartate aminotransferase {EC 2.6.1.1} structural gene, aspC, of Escherichia coli K-12 was determined . The coding region of the aspC gene contained 1,188 nucleotide residues and encoded 396 amino acid residues . The amino acid sequence deduced from the nucleotide sequence agreed perfectly with that of the protein recently determined for the aspartate aminotransferase of E . coli B (Kondo, K., Wakabayashi, S., Yagi, T., & Kagamiyama, H . (1984) Biochem . Biophys . Res . Commun . 122, 62-67). EMBO J, 1985 Apr, 4(4), 1007 - 11 Cloning and expression in Escherichia coli of a surface antigen of Plasmodium falciparum merozoites; Cheung A et al.; A complementary DNA library was constructed from mRNA purified from asexual blood forms of Plasmodium falciparum . Among the members of this library we have identified a plasmid (pMC31-1) coding for a polypeptide exposed at the surface of merozoites, the invasive stage of the asexual cycle . This plasmid was identified by direct expression using both polyclonal and monoclonal antibodies specific for a schizont polypeptide of 200 kd which has been shown to be processed to an 83-kd polypeptide expressed at the surface of merozoites . The cDNA portion of the pMC31-1 plasmid hybridizes with DNA from three isolates of P . falciparum . Antisera raised against extracts of Escherichia coli harbouring pMC31-1 react with surface and internal structures of schizonts and with the surface of merozoites from all the isolates of P . falciparum examined . These results suggest that plasmid pMC31-1 encodes an antigen of value for the development of a vaccine against malaria. Microbiologica, 1985 Apr, 8(2), 211 - 5 Nontoxicity of an oil shale process water to Escherichia coli; Adams JC; The survival of Escherichia coli in the presence of an oil shale process water was studied over a five day period . The organism survived better in the presence of one or ten percent concentration of the process water than it did in distilled or tap water . Water chemistry of the diluent appeared to be important to the survival of the organism. Microbiologica, 1985 Apr, 8(2), 181 - 90 Modifications of surface properties in some enteropathogenic serogroups of Escherichia coli; Tufano MA et al.; Studies have been carried out on protein and fatty acid patterns present in the outer layers of E . coli strains which are considered pathogenic in relation to their serogroup . The patterns were related to surface properties of hydrophobicity of the same cells and to their resistance to phagocytosis . The results obtained demonstrate the presence in the pathogenic serogroups of a proteic band of high molecular weight as well as differences in the percentages of fatty acids with respect to the non-pathogenic serogroups . Furthermore, these serogroups are more resistant to phagocytosis; this resistance seems to be correlated to a greater surface hydrophilia of the pathogenic serogroups, leading to different relations of the lipidic and protein fraction of the outer layers. Bioorg Khim, 1985 Apr, 11(4), 480 - 91 {Topography of cysteine residues in DNA-dependent RNA-polymerase}; Seliuchenko OA et al.; Chemical modification of DNA-dependent RNA polymerase with monoiod-{14C}acetic acid and N-{14C}ethylmaleimide has been carried out and position of superficial and functionally important as well as enzymatically non-significant, exposed cysteine residues have been localised in the enzyme . Topography of cysteine residues in the RNA-polymerase alpha-subunit is thoroughly studied . The results are summarized in a model. Arch Microbiol, 1985 Apr, 141(3), 209 - 13 Ethylene formation by cultures of Escherichia coli; Ince JE et al.; Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH4Cl and methionine resulted in production of ethylene into the culture headspace . When methionine was excluded from the medium there was little formation of ethylene . Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth . Ethylene formation was stimulated by increasing the medium concentration of Fe3+ when it was chelated to EDTA . Lowering the medium phosphate concentration also appeared to stimulate ethylene formation . Ethylene formation was inhibited in cultures where NH4Cl remained in the stationary phase . Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine . Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation . It is concluded that ethylene production by E . coli exhibits the typical properties of secondary metabolism. Appl Environ Microbiol, 1985 Apr, 49(4), 1016 - 8 IncH plasmids in Escherichia coli strains isolated from broiler chicken carcasses; Chaslus-Dancla E et al.; Plasmids conferring tellurite resistance were transferred at low temperature (27 degrees C) from Escherichia coli strains isolated from chicken carcasses at the time of slaughter and after storage . They belonged to group IncH, as evidenced by their large molecular weight and incompatibility with plasmid pIP233 . E . coli strains contaminating chickens meat can thus represent a source of IncH plasmids in the food chain of humans. Scand J Gastroenterol, 1985 Apr, 20(3), 279 - 84 Effect of intravenous and intraduodenal administration of Escherichia coli endotoxin on the porcine pancreas as evaluated by changes in the serum cationic trypsin-like immunoreactivity; Florholmen J et al.; In six pigs intravenous administration of Escherichia coli endotoxin caused septic shock and a significant increase in serum cationic trypsin-like immunoreactivity (CTLI), with activation of cationic trypsinogen to trypsin and formation of complexes between cationic trypsin, on the one hand, and alpha 2-macroglobulin and alpha 1-antitrypsin, on the other, compatible with acute pancreatitis . In contrast, intraduodenal infusion of E . coli endotoxin to seven other pigs was without effect on the general circulation and on the serum CTLI. DNA, 1985 Apr, 4(2), 139 - 46 Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNA; Jacobs P et al.; A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase . This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem . The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1) . The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone . A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2 . Heat induction of E . coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase. DNA, 1985 Apr, 4(2), 115 - 26 Construction and characterization of a library of metallothionein coding sequence mutants; Pine R et al.; New possibilities for experimental investigation of the metallothionein system at the DNA, RNA, and protein levels are now available via a library of metallothionein coding sequence mutants . Appropriate treatment in vitro with sodium bisulfite of the sense or antisense DNA strands in M13 phage vectors and propagation in Escherichia coli BD1528 (ung-) produced two unique collections in the library containing either C to T or G to A transition mutations in the codons at low and high frequencies . The strategy for mutagenesis takes advantage of degeneracy in the genetic code so that no cysteine codons are replaced in the CT mutants while all are subject to change in the GA collection . Two hundred and sixty-four clones from the library have been sequenced and G to A transitions affecting each of the 20 cysteine codons in metallothionein have been detected . Other mutations in codons for amino acids proposed to be important for metallothionein function have also been identified. Anal Biochem, 1985 Apr, 146(1), 180 - 3 Lysine decarboxylase assay by the pH-stat method; Vienozinskiene J et al.; A simple assay of lysine decarboxylase is described . It is based on measuring the rate of titration of OH- ions, released during decarboxylation with the aid of a pH-stat apparatus . The continuous recording of the reaction progress may be easily performed . Using the pH-stat method the Km for lysine decarboxylase from E . coli is 2.0 mM. Anal Biochem, 1985 Apr, 146(1), 150 - 7 The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase; Hunt JB et al.; The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol . The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex . Using greater than 10-fold PAR to free Zn2+ at pH 7.0, delta epsilon = 6.6 +/- 0.2 X 10(4) M-1 cm-1 at 500 nm (20 degrees C) for (PAR)2Zn2+ complex formation (beta'2 approximately equal to 10(12) M-1) . In kinetic studies at pH 7.0, PAR (10(-4) M) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 X 10(7) M-1 s-1 for forming the 1:1 PAR:Zn2+ complex . These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins. Acta Physiol Scand, 1985 Apr, 123(4), 399 - 404 The origin and fall of plasma motilin during Escherichia coli endotoxin shock in pigs; Jenssen TG et al.; Plasma motilin decreased significantly during a 120 min intravenous Escherichia coli endotoxin infusion which induced deep shock in six anaesthetized pigs . Plasma motilin was significantly higher in the portal vein and significantly lower in the internal jugular vein than in the aorta and the superior caval vein . Following gastrointestinal resection with blood sampling from the portal vein, the aorta and the superior caval vein in another six pigs, motilin almost disappeared in non-portal plasma, while it decreased significantly in portal plasma in which it also fell further during E . coli endotoxin infusion . The results support the opinion that the gastrointestinal tract is the main source of plasma motilin. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2518 - 22 Expression of Plasmodium falciparum surface antigens in Escherichia coli; Ardeshir F et al.; The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity . Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle . With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P . falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8 . Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P . falciparum, and 95 clones expressing parasite antigens were identified . Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays . Several different P . falciparum antigens were identified by these assays . Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface . Thus, we have identified cDNA clones to three different P . falciparum antigens that may be important in protective immunity. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2354 - 8 Expression in Escherichia coli of a fusion protein product containing a region of the adenovirus DNA polymerase; Rekosh D et al.; The bulk of an open reading frame extending from map coordinates 23.3 to 14.2 in region E2b of the adenoviral genome has been cloned and expressed from a chimeric plasmid in Escherichia coli . The cloning strategy used created a fusion protein of 124,000 daltons, which contained greater than 98% adenovirus-encoded sequences . Antiserum raised against this protein reacted with the authentic 140,000-dalton adenovirus DNA polymerase . Another serum raised against a synthetic hexapeptide whose sequence corresponded to the predicted carboxyl terminus of adenovirus-encoded DNA polymerase also reacted with the fusion protein and authentic adenovirus DNA polymerase . These results demonstrate that the cloned region of DNA encodes the adenovirus DNA polymerase. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2325 - 9 Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I; O'Connor D et al.; Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment . Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation . By a combination of primers and "nick-mers", conditions of single-strand-directed DNA synthesis and nick-translation could be created . Our results show that the polymerase can bypass both types of lesions . Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template . Only purines, with guanine more favored than adenine, are incorporated across both lesions . Hesitation during bypass could not be detected . The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates . They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2301 - 5 Deletions within a hinge region of a specific DNA-binding protein; Little JW et al.; Many proteins are organized as a set of compact functional domains connected by flexible, exposed segments of the polypeptide chain . To study one of these connector regions, we isolated a series of functional in-frame deletions in the central portion of a specific DNA-binding protein, the LexA repressor of Escherichia coli . These mutant proteins fell into two main classes: those with small deletions of two to eight amino acids functioned as repressor about as well as did wild type, while those with large deletions of 17-22 amino acids functioned well only at considerably higher concentrations . The mutant proteins were resistant to the specific cleavage reaction that triggers the SOS response . These data suggest that the conformation of the hinge region in LexA protein is important for cleavage . By contrast, the hinge plays a topological role in repressor function, connecting the two functional halves of the protein; in the SOS response, this function of the hinge is inactivated by cleavage, leading to inactivation of the repressor. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2272 - 6 Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme; Straus D et al.; We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis . Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E . coli isomerase . The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6 . With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher . The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M . By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself. J Gen Microbiol, 1985 Apr, 131 ( Pt 4), 939 - 43 Inducible plasmid-mediated copper resistance in Escherichia coli; Rouch D et al.; The copper resistance in Escherichia coli determined by plasmid pRJ1004 is inducible . The level of resistance is proportional to the inducing dose of copper . The level of copper resistance in induced and uninduced cells changes with the growth phase of the culture . Induced resistant cells accumulate less copper than uninduced cells, so that reduced accumulation may be the mechanism of resistance . We propose that the inducible plasmid-coded copper resistance interacts with the normal metabolism of the cell to protect against toxic levels of copper while allowing continued operation of copper-dependent functions. J Clin Microbiol, 1985 Apr, 21(4), 630 - 3 Survey in Vanuatu on enterotoxigenic Escherichia coli in children and infants with and without acute diarrhea; Germani Y et al.; We have studied the incidence of enterotoxigenic Escherichia coli (ETEC) strains isolated from infants with and without diarrheal diseases in Vanuatu, South Pacific . Over a period of 5 months we have isolated enterotoxigenic E . coli strains from 29 (26.6%) of 109 children with acute diarrhea and from 13 (21.6%) of 60 children of the control group . In the group with diarrhea, 7 (6.4%) strains released heat-labile toxin, 7 (6.4%) released heat-stable toxin, and 15 (13.7%) produced both heat-labile and heat-stable toxin . In the control group, only one strain (1.6%) produced heat-stable toxin, 12 (20%) produced heat-labile toxin, and none produced both . Association of strains releasing heat-stable toxin or both heat-labile and heat-stable toxin with diarrhea was highly significant as shown by statistical analysis . The O serogroups and colonization factors CFA/I and CFA/II are presented. Cell, 1985 Apr, 40(4), 767 - 74 Specific DNA binding of GAL4, a positive regulatory protein of yeast; Giniger E et al.; We show by the following series of experiments that the yeast positive regulatory protein GAL4 binds to four sites in the upstream activating sequence UASG to activate transcription of the adjacent GAL1 and GAL10 genes . GAL4 protein expressed in E . coli protected guanine residues in UASG from methylation by dimethyl sulfate . The same set of protections was seen in vivo in yeast and depended on the GAL4+ allele . This protection pattern is consistent with the idea that GAL4 protein binds to four related 17 bp sequences, each of which displays approximate 2-fold rotational symmetry . A single near-consensus synthetic 17 bp oligonucleotide, installed in front of the yeast GAL1 or CYC1 transcription units, conferred a high level of galactose inducibility upon these genes . Further experiments suggest that one mechanism of glucose repression is inhibition of the binding of GAL4 protein to DNA. Biochem Pharmacol, 1985 Apr 1, 34(7), 1065 - 71 Photoaffinity labeling of the tetracycline binding site of the Escherichia coli ribosome . The uses of a high intensity light source and of radioactive sancycline derivatives; Hasan T et al.; {3H}Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome . However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity . In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of {3H}TC with a common, rather low intensity, lamp . On the basis of the results of this study we next explored the usefulness for photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself . Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site. Gan To Kagaku Ryoho, 1985 Apr, 12(4), 789 - 97 {A carcinogens--methods for detecting environmental muta/carcinogens}; Morimoto K et al.; The major causes of cancer in the developed countries are now thought to be environmental or man-made factors . Because most carcinogens are also mutagens, the detection and identification of muta/carcinogens to humans seem to be very important in the environmental sciences . We describe here, i) in vitro and in vivo short-term assays for muta/carcinogens in the environment, ii) possible method for monitoring human exposure to muta/carcinogens, and iii) some trials for estimating human risk for the development of cancers due to environmental mutagens. Am J Surg, 1985 Apr, 149(4), 487 - 94 Intrahepatic pyogenic abscesses: treatment by percutaneous drainage; Gerzof SG et al.; During a 6 year period, 18 liver abscesses in 12 patients were identified by computerized tomography . Five patients had presumed hematogenous seeding . Five patients previously had bilioenteric anastomoses, stents, or both to relieve obstructive jaundice . Four patients with abscesses had recent abdominal operations . Diagnosis was established by guided needle aspiration and treatment was provided by percutaneous catheter drainage . Organism-specific antibiotics were administered to all patients . Patients were evaluated for recurrence by serial computerized tomographic studies and were clinically followed up for a minimum of 15 months . Ten of 12 patients (83 percent) and 16 of 18 abscesses (89 percent) were successfully treated by percutaneous catheter drainage . Two failures required operative intervention . In summary, the low morbidity and high success rate in treating hepatic abscesses by percutaneous drainage suggests that this therapy be tried before operative intervention is considered. Am J Physiol, 1985 Apr, 248(4 Pt 2), R471 - 8 Glucose kinetics and body temperature after lethal and nonlethal doses of endotoxin; Lang CH et al.; Rats were injected with doses of endotoxin ranging from 1,000 {lethal dose approximately 50% (LD50)} to 0.01 microgram/100 g, and alterations in hemodynamics, glucose kinetics, and body temperature were studied over the subsequent 4 h . Doses of 10 micrograms/100 g or less were consistently nonlethal over 72 h . Decreases in arterial blood pressure and cardiac output were evident in rats receiving 1,000-10 micrograms/100 g endotoxin . Doses of endotoxin between 1,000 and 10 micrograms produced an early hyperlactacidemia evident by 1 h, whereas the lower doses (1 and 0.1 microgram) induced elevations that exhibited a delayed temporal response . The rates of glucose appearance (Ra) and disappearance (Rd) were increased early and transiently by the higher doses of endotoxin . Lower doses increased glucose Ra and Rd between 2 and 4 h after endotoxin . A febrile response was elicited by 10, 1, and 0.1 microgram/100 g endotoxin, while hypothermia was seen in animals receiving higher doses . Thus high doses of endotoxin induced metabolic and hemodynamic alterations that were temporally associated . Very low nonlethal doses of endotoxin (up to 4 orders of magnitude less than LD50) induced metabolic changes that appeared to be independent of hemodynamic disturbances but were temporally associated with the observed hyperthermia. Am J Physiol, 1985 Apr, 248(4 Pt 1), E420 - 4 Endotoxin increases vasopressin release independently of known physiological stimuli; Kasting NW et al.; Plasma vasopressin (AVP) increases after endotoxin administration in freely behaving unanesthetized rats . The present experiments sought to determine the factors that mediate this vasopressin response . Endotoxin (150 micrograms/kg iv) elicited a significant increase in plasma AVP concentration . This response was accompanied by unchanged plasma osmolality, hypotension, increased hematocrit (reflecting decreased plasma volume), hypothermia, and hyperglycemia . Pretreatment with the prostaglandin synthesis inhibitor, indomethacin (5 mg/kg sc), had no effect on the vasopressin response to endotoxin but abolished or significantly attenuated the changes in blood pressure, hematocrit, temperature, and plasma glucose while leaving plasma osmolality unaltered . These investigations indicate that endotoxin stimulates vasopressin secretion into plasma independently of changes in plasma osmolality, systemic blood pressure, plasma volume, body temperature, or plasma glucose . The results also suggest that vasopressin responses to endotoxin are not mediated by prostaglandins, whereas prostaglandins do play a role in endotoxin's effects on blood pressure, plasma volume, temperature, and plasma glucose. Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2014 - 8 Chicken triosephosphate isomerase complements an Escherichia coli deficiency; Straus D et al.; We present the sequence of full-length chicken triosephosphate isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) mRNA based on the analysis of cDNA and genomic clones . To isolate cDNA clones encoding the enzyme, we screened a muscle cDNA library with radioactively labeled cDNA made from RNA that had been enriched by immunoselection of polysomes . We blocked the signal caused by contaminating species in the probe with cloned DNA corresponding to the contaminants . Screening a chicken genomic library with cDNA coding for triosephosphate isomerase led to the isolation of phage containing the entire gene, which we used to map the transcriptional start . When placed downstream from a hybrid trp-lac promoter, the cDNA encoding the chicken enzyme programs the synthesis of functional protein, as judged by enzymatic criteria and by complementation of an Escherichia coli mutant that is deficient in bacterial triosephosphate isomerase. Mutat Res, 1985 Apr, 142(4), 163 - 7 Mutagenicity of nitropyrenes for Escherichia coli: requirement for increased cellular permeability; McCoy EC et al.; Nitropyrenes are mutagenic to E . coli strains that have increased permeabilities to large molecules and carry plasmid pKM101. J Bacteriol, 1985 Apr, 162(1), 5 - 8 Localization of acyl carrier protein in Escherichia coli; Jackowski S et al.; Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli . These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H . Van den Bosch, J.R . Williamson, and P.R . Vagelos, Nature {London} 228:338-341, 1970) . Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro . A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane. J Bacteriol, 1985 Apr, 162(1), 451 - 3 Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise; Szupica CJ et al.; Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise . Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes. J Bacteriol, 1985 Apr, 162(1), 448 - 50 Glutathione reductase is not required for maintenance of reduced glutathione in Escherichia coli K-12; Tuggle CK et al.; Seven independently isolated glutathione reductase-deficient (gor) Escherichia coli mutants were found to have an in vivo glutathione redox state that did not significantly differ from that of the parental strain, 98 to 99% reduced . Strains containing both a gor mutation and either a trxA mutation (thioredoxin deficient) or a trxB mutation (thioredoxin reductase deficient) were able to maintain a 94 to 96% reduced glutathione pool, suggesting that glutathione can be reduced independently of glutathione reductase and thioredoxin reductase. J Bacteriol, 1985 Apr, 162(1), 374 - 82 Duplication of Escherichia coli during inhibition of net phospholipid synthesis; Pierucci O et al.; In Escherichia coli BB26-36, the inhibition of net phospholipid synthesis during glycerol starvation affected cell duplication in a manner that was similar in some respects to that observed during the inhibition of protein synthesis . Ongoing rounds of chromosome replication continued, and cells in the D period divided . The initiation of new rounds of chromosome replication and division of cells in the C period were inhibited . Unlike the inhibition of protein synthesis, however, the accumulation of initiation potential in dnaA and dnaC mutants at the nonpermissive temperature was not affected by the inhibition of phospholipid synthesis . Furthermore, proteins synthesized during the inhibition of phospholipid synthesis can be utilized later for division . The results are consistent with a dual requirement for protein and phospholipid synthesis for both the inauguration of new rounds of chromosome replication and the initiation of septum formation . Once initiated, both processes progress to completion independent of continuous phospholipid and protein synthesis. J Bacteriol, 1985 Apr, 162(1), 367 - 73 Cloning and expression of the transhydrogenase gene of Escherichia coli; Clarke DM et al.; Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank . The three plasmids were subjected to restriction endonuclease analysis . A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13 . Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment . This segment was located at 35.4 min in the E . coli genome . Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase . The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids . Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11 . Both polypeptides were required for expression of transhydrogenase activity. J Bacteriol, 1985 Apr, 162(1), 353 - 60 Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli; Sankar P et al.; Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity . The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322 . The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity . A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA . This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase . Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128 . Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase . This inhibition occurred even in a prototrophic E . coli, strain K-10, but only during an early induction period . These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase . For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed . All four genes map between 58 and 59 min in the E . coli chromosome. J Bacteriol, 1985 Apr, 162(1), 344 - 52 Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism; Lee JH et al.; A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli . Mutant strains isolated by this procedure can be divided into two major classes . Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor . Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities . All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase . The mutation in class I mutants mapped near 65 min of the E . coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome . The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl . These results indicate that there are two hyd operons and one hup operon in the E . coli chromosome . The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell. J Bacteriol, 1985 Apr, 162(1), 29 - 34 General recombination in Escherichia coli K-12: in vivo role of RecBC enzyme; Yancey SD et al.; Heterozygous lacZ- merodiploids of Escherichia coli K-12 have been used to study the role of the RecBC enzyme in general recombination . The transcribable intermediate assay detects the product of early steps in recombination without requiring the formation of viable recombinant colonies . Recombination is initiated by infection with lambda precA+ . We have found that transcribable intermediate formation in crosses between F42 lac and chromosomal lac is dependent on F fertility functions and an active RecBC enzyme . Thus, the products of the recB and recC genes are required in early steps of recombination between these two substrates . Introduction of the F42 lac donor DNA by conjugation immediately after infection with lambda precA+ abolishes the requirement for an active RecBC enzyme. J Bacteriol, 1985 Apr, 162(1), 242 - 7 Amount and chain length of polyphosphates in Escherichia coli depend on cell growth conditions; Rao NN et al.; Anaerobiosis induced an accumulation of polyphosphates (poly Pi) in a phosphate-rich medium by an alkaline-phosphatase constitutive mutant of Escherichia coli . The total poly Pi content was maximum at around 6 h of anaerobic growth . Both trichloroacetic acid- and NaOH-soluble poly Pi were found to be present . The acid-soluble fraction consisted mainly of a linear polymer of about 20 +/- 5 phosphate units, whereas the alkali-extractable poly Pi fraction contained at least four molecular species of higher chain length as determined by gel filtration . The majority of poly Pi extracted at 6 h had lower chain lengths than those extracted from cells incubated for 24 h . In vivo 31P nuclear magnetic resonance spectra of E . coli cells as a function of growth conditions were consistent with the in vitro extract results. J Bacteriol, 1985 Apr, 162(1), 190 - 5 Strain-specific chemotaxis of Azospirillum spp; Reinhold B et al.; Chemotactic responses of three Azospirillum strains originating from different host plants were compared to examine the possible role of chemotaxis in the adaptation of these bacteria to their respective hosts . The chemotaxis to several sugars, amino acids, and organic acids was determined qualitatively by an agar plate assay and quantitatively by a channeled-chamber technique . High chemotactic ratios, up to 40, were obtained with the latter technique . The chemotactic response did not rely upon the ability of the bacteria to metabolize the attractant . Rather, it depended on the attractant concentration and stereoconfiguration . Chemotaxis was found to be strain specific . Differences were particularly observed between a wheat isolate and strains originating from the C4-pathway plants maize and Leptochloa fusca . In contrast to the other two strains, the wheat isolate was strongly attracted to D-fructose, L-aspartate, citrate, and oxalate . The other strains showed maximal attraction to L-malate . The chemotactic responses to organic acids partially correlate with the exudation of these acids by the respective host plants . Additionally, a heat-labile, high-molecular-weight attractant was found in the root exudates of L . fusca, which specifically attracted the homologous Azospirillum strain . It is proposed that strain-specific chemotaxis probably reflects an adaptation of Azospirillum spp . to the conditions provided by the host plant and contributes to the initiation of the association process. J Bacteriol, 1985 Apr, 162(1), 106 - 9 Escherichia coli mutants possessing an Li+-resistant melibiose carrier; Shiota S et al.; Escherichia coli K-12 strains in the absence of the lactose carrier grew on the disaccharide melibiose as the sole source of carbon . The presence of 0.1 mM Li+ in the medium strongly inhibited growth of such cells, and Li+-resistant mutants appeared after several days of incubation . These mutants showed altered cation coupling to melibiose transport via the melibiose carrier . Cotransport between H+ and melibiose was lost in the mutants, although Na+-melibiose cotransport was retained . We observed no Li+-melibiose cotransport . Therefore, these mutants represent a new type of cation-coupling mutants of the melibiose carrier. Infect Immun, 1985 Apr, 48(1), 73 - 7 Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin; Leong J et al.; The nucleotide sequence of the LT-BH cistron (eltBH) from an enterotoxigenic Escherichia coli strain infectious for humans was determined and compared with the LT-B cistron sequence from a porcine E . coli isolate . Both cistrons were shown to comprise 375 nucleotide base pairs, and discrepancies were detected at eight positions . Of the nonhomologous base pairs, six resulted in codon changes that would lead to amino acid variations . The nucleotide sequence distal to both LT-B cistrons was also determined, and only three differences were detected in 197 base pairs . An HhaI site unique to eltBH was shown to be present in all the heat-labile (LT) genes from 31 human isolates surveyed, whereas the restriction enzyme recognition site was absent in the gene from 46 porcine E . coli isolates . The results suggest that two genetically discernable LT groups are identifiable and that the groups are also distinguishable by the isolation source (human or porcine) of the infecting E . coli strains. Radiology, 1985 Apr, 155(1), 101 - 4 Adrenal abscess in the neonate; Atkinson GO Jr et al.; Adrenal abscess in the neonate is a rare complication of adrenal hemorrhage . The radiographic and clinical findings of 12 previously published cases and two new cases of adrenal abscess in the newborn are presented . Sonography was the most helpful examination in distinguishing a suprarenal lesion from an intrarenal lesion and in demonstrating the morphology of the abscess. J Infect Dis, 1985 Apr, 151(4), 716 - 20 A multistate outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli in imported semisoft cheese; MacDonald KL et al.; In September 1983, three clusters of gastrointestinal illness with similar symptoms affected 45 persons in Washington, D.C., after office parties . The illness lasted a mean of 4.4 days and was characterized by watery diarrhea (91%), abdominal cramps (80%), headache (38%), nausea (38%), and subjective fever (20%) . Illness was strongly associated with having eaten imported French Brie cheese one to six days before onset of illness (P less than .0001 by Fisher's two-tailed exact test) . After publicity about these outbreaks, additional cheese-associated cases were identified over an eight-week period in Illinois, Wisconsin, Georgia, and Colorado . Stool specimens from ill persons in four states yielded Escherichia coli serotype O27:H20 . These organisms produced heat-stable enterotoxin and had similar plasmid profiles . When commercially distributed foods are contaminated, enterotoxigenic E . coli can cause widespread disease even in a developed country, and the disease can easily escape correct diagnosis. Am J Vet Res, 1985 Apr, 46(4), 821 - 30 Experimental porcine eperythrozoonosis: T-lymphocyte suppression and misdirected immune responses; Zachary JF et al.; Immune responses and hematologic alterations were investigated in splenectomized pigs after IM inoculation with Eperythrozoon suis . Early hematologic alterations were massive parasitism of RBC, severe hypoglycemia, moderate bilirubinemia, and mild anemia; later findings included severe anemia, minimal parasitism of RBC, spontaneous agglutination of RBC at 25 C and 4 C which was reversible at 37 C, transient thrombocytopenia, and mild bilirubinemia . The humoral immune responses consisting of a transitory hyperglobulinemia and increase of indirect hemagglutination (IHA) titers against E suis were attributed to immunoglobulin M cold agglutinins . Cell-mediated immune responses, measured by phytohemagglutinin- and pokeweed mitogen-induced lymphocyte blastogenesis, were reduced after massive parasitemia . Blastogenesis induced by Escherichia coli lipopolysaccharide mitogen was increased before the hyperglobulinemia and an increase in IHA titer . There was an increase in the uptake of {3H}thymidine by lymphocytes cultured without mitogens after the decline in total globulin concentration and IHA titer. Cell Immunol, 1985 Apr 1, 91(2), 520 - 7 Effect of lipopolysaccharide on the stimulation of macrophage Fc-dependent phagocytosis by splenic B lymphocytes; Coleman DL et al.; Macrophage phagocytic activity is regulated by a variety of products derived from activated lymphocytes . It has been reported that nonactivated splenic B and T lymphocytes enhance macrophage glucose metabolism . In addition, the enhancement of macrophage glucose metabolism was further increased by direct effects of bacterial lipopolysaccharide (LPS) on B, but not T, lymphocytes . In the present study, the effect of purified murine splenic B and T lymphocytes on Fc-dependent phagocytosis by thioglycollate-elicited peritoneal macrophages in the presence or absence of LPS has been investigated . Fc-dependent phagocytosis was assayed by measuring the ingestion of 51Cr-tagged sheep erythrocytes . After 3 or 4 days in culture, nonadherent spleen cells (NASC) and B and T lymphocytes from C3H/HeN (LPS-responder) mice produced 92 +/- 27%, 83 +/- 13%, and 147 +/- 33% increases in C3H/HeJ (LPS-hyporesponder) macrophage phagocytic activity, respectively . A similar effect was observed in Balb/c mice . Cell-free supernatant from NASC and B lymphocytes precultured for 2 or 4 days produced a 74 +/- 20% and 157 +/- 42% increase in phagocytosis respectively . At concentrations which have been previously shown to markedly enhance the ability of splenic B lymphocytes to stimulate macrophage glucose metabolism, Escherichia coli K235 LPS (10 micrograms/ml) did not alter the stimulatory effects of any of the splenic lymphocyte populations on macrophage Fc-dependent phagocytosis . These data suggest that B lymphocytes produce a soluble factor(s) which stimulates macrophage phagocytosis . In addition, LPS has different effects on the regulation of macrophage phagocytic activity and metabolism by B lymphocytes. Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2024 - 8 Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation; Harada N et al.; By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice . mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro . Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B . Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes . The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I . 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice . This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females . Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene. DNA, 1985 Apr, 4(2), 147 - 56 The primary structure of the imported mitochondrial protein, ornithine transcarbamylase from rat liver: mRNA levels during ontogeny; McIntyre P et al.; Ornithine transcarbamylase, one of the enzymes of the urea cycle in ureotelic organisms, is synthesized in the cytoplasm of hepatocytes as a precursor larger than the mature form found in the mitochondrial matrix . We deduced the amino acid sequence of the precursor of ornithine transcarbamylase from rat liver from the nucleotide sequence of overlapping cDNA clones spanning the complete coding region, 3' untranslated region, and most of the 5' untranslated region of the mRNA . The mature enzyme consists of 322 amino acids and is derived from the larger precursor by proteolytic removal of 32 amino acids from the amino-terminus . The amino-terminal extension contains eight basic and no acidic residues . This highly basic character appears to be a feature of presequences on cytoplasmically synthesized mitochondrial proteins . Comparison of the amino acid sequence determined for the enzyme from rat with that from human liver (Horwich et al., 1984) shows that there is a high degree of homology between the sequences of the mature protein (93%) and relatively less homology between the sequences of the amino-terminal extension (72%) . The ornithine transcarbamylase from rat liver also shows a considerable degree of amino acid homology (44%) with the enzyme from Escherichia coli (Van Vliet et al., 1984) and leads to suggestions about residues involved in substrate binding and catalysis . An analysis of levels of RNA in fetal and neonatal liver shows that ornithine transcarbamylase mRNA levels increase from about 40% of adult levels at day 14 of gestation to a peak at day 20 of gestation, and, after a drop around the time of birth, rises to adult levels during the second week after birth. Genetika, 1985 Apr, 21(4), 673 - 5 {Isolation of Escherichia coli K-12 mutants that affect the development of phage phi m173 (imm phi 80)}; Odoevskaia ER et al.; Escherichia coli mutants which block the development of a number of lambdoid phages, particularly, phi m173 and phi 80 were selected . These mutants have different phenotypes, being resistant to different groups of lambdoid phages . There are also differences between new mutants and gro mutants of E . coli studied earlier . The results obtained support the suggestion that no replication of different lambdoid phages takes place in these mutants. Genetika, 1985 Apr, 21(4), 530 - 40 {Formation and genetic structure of polylysogens for lambda and phi 80 and their lambda att80 hybrid infecting wild-type Escherichia coli}; Kholodii GIa et al.; The frequency of polylysogeny and the genetic structure of polylysogens were studied for phages lambda, phi 80 and lambda att80 . For none of these phages does frequency of polylysogeny vary by more than a factor of 2 within a wide range of multiplicities of infection (from 10(-3) up to 10) but the relative location of the prophages on the host chromosome is different . In the case of lambda, polylysogens are formed with a high frequency (0.20-0.41) and the prophages are inserted in tandem into the primary (normal) att site . In the case of phi 80 and lambda att80, polylysogens occur about 10 times less frequently and usually have one prophage inserted into the primary attachment site and another (sometimes, also a third) in one of the secondary ones . Wild-type Escherichia coli was shown to possess at least four secondary att80 sites, two of which (close to the his and tolC loci) are preferred . The frequency of secondary integration of phi 80 and lambda att80 does not differ significantly in the wild-type host and in cells deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at MOI 10) . Certain properties of the phi 80 lysogens make it more difficult to decode their genetic structure. Mutat Res, 1985 Apr-May, 156(1-2), 69 - 75 Induction of lambda prophage by furazolidone; Pal AK et al.; A dose-dependent prophage induction by furazolidone exhibited a gradual rise to a maximum, corresponding to an exposure dose of 1.2 microgram/ml X h and a gradual fall thereafter . A 2-3-fold higher level of induction was achieved when the lysogens were treated with furazolidone in the presence of a metabolizing mixture . A maximum of about 70% efficiency of induction was achieved . Kinetics of prophage induction by any concentration of furazolidone exhibited a common pattern, viz., an initial rise for 15-20 min, then a plateau extending up to about 60 min and a faster rise thereafter . Higher concentrations of the drug (10 micrograms/ml) exhibited a toxic effect . Chloramphenicol at a concentration of 20 micrograms/ml inhibited the furazolidone-induced prophage induction, the plaque-forming units gradually decreasing from several minutes after the chloramphenicol treatment . The burst size of the lysogens was not significantly affected by treatment with 2 micrograms/ml of furazolidone up to a period of about 10 min, but thereafter, decreased faster with the duration of furazolidone treatment . The "latent period' of induction decreased linearly with the duration of furazolidone treatment. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2339 - 43 NH2-terminal arm of phage lambda repressor contributes energy and specificity to repressor binding and determines the effects of operator mutations; Eliason JL et al.; Several lines of evidence indicate that the phage lambda repressor recognizes its operator by using, in part, an alpha helix (the "recognition helix"), which it inserts into the major groove of DNA . In addition to its recognition helix, lambda repressor has an "arm," consisting of the first six amino acids, that wraps around the DNA helix . We constructed plasmids that, in Escherichia coli, direct the expression of derivatives of lambda repressor that lack the NH2-terminal one, three, six, or seven amino acids . We studied these modified proteins in vivo and in vitro, and from our results we argue that the arm: contributes a large portion of the binding energy; helps to determine sequence specificity of binding and, in particular, the relative affinities for two wild-type binding sites; determines entirely repressor's response to one operator mutation (a "back-side" mutation); magnifies repressor's response to other operator mutations ("front-side" mutations); and increases the sensitivity of repressor binding to salt concentration and temperature. J Bacteriol, 1985 Apr, 162(1), 72 - 7 Copy mutant of mini-Rts1: lowered binding affinity of mutated RepA protein to direct repeats; Terawaki Y et al.; Nucleotide sequence analysis of mini-Rts1 and its copy mutant disclosed the presence of two clusters of direct-repeat sequences flanking the coding region for the 33,000-dalton RepA protein and two base substitutions on the mini-Rts1cop1 genome (Kamio et al., J . Bacteriol . 158:307-312, 1984) . On subcloning of the left cluster (incI) that is located downstream from repA, the five 24-base-pair repeats expressed a stronger incompatibility toward mini-Rts1 than did the four repeats . The right cluster (incII) that contains three 21-base-pair repeats also exhibited strong incompatibility toward mini-Rts1 . By separating the two base substitutions of mini-Rts1cop1, the mutation that is responsible for the copy increase was determined to be a single base change in the RepA coding region . Both clusters of the repeats, cloned separately into the vector plasmid, showed a weaker incompatibility toward mini-Rts1cop1 than to the wild-type mini-Rts1 . These findings suggest a lowered binding affinity of the mutated RepA protein to the direct repeats. Mol Gen Mikrobiol Virusol, 1985 Apr, (4), 35 - 9 {Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction}; Elizbarashvili DA et al.; The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation . Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell . Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30% . Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001 . The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells . Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants . The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA. Mol Gen Mikrobiol Virusol, 1985 Apr, (4), 26 - 30 {Molecular cloning of provirus sequences of Rauscher leukemia virus from mouse erythroleukemia cell genome}; Neznanov NS et al.; Provirus from a component of Rauscher leukaemia virus (RLV) has been cloned . The provirus (the size of 5000 b . p.) contains two LTR sequences and shares expressed sequence homology with Mo-MuLV . Restriction analysis and determination of the LTR nucleotide sequence and of the site from 3'-end of proviral genome have shown the cloned provirus to be the SFEV component of RLV . LTR from this cloned provirus contains all sites necessary for transcription: CAAT and TATA sequences, "cap" site and polyadenylation signal . The LTR of the cloned provirus from SFEV component of RLV has been shown to function as a promoter in E . coli cells. Proc Natl Sci Counc Repub China B, 1985 Apr, 9(2), 110 - 8 Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan; Lee YH et al.; The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E . coli JM103 host . Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method . The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site . Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found . Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence . The physical mapping of HBV DNA was performed with several restriction endonucleases . Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases . The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes. EMBO J, 1985 Apr, 4(4), 1059 - 66 Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene; Bech FW et al.; Escherichia coli relB mutants react to amino acid starvation by several abnormal responses, including accumulation of a translational inhibitor . We have isolated a relB-complementing plasmid from the Clarke and Carbon E . coli DNA library . From this plasmid we sequenced a 2140-bp segment which included the relB gene by the following two criteria: (i) it complements chromosomal relB mutations, (ii) the corresponding DNA segment cloned from chromosomal DNA of three relB mutants was defective in relB complementation . All three mutations fell within an open reading frame of 79 amino acids . A polypeptide of 9 kd compatible with this open reading frame was synthesized in maxicells and is in all probability the product of the relB gene . By nuclease S1 mapping we have determined the transcription start and stop of an 870 base transcript of the relB gene. EMBO J, 1985 Apr, 4(4), 1053 - 8 Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control; Mizushima-Sugano J et al.; We have located the DNA sequence involved in the stringent control of the Escherichia coli tufB operon . Various deletion and insertion mutants of the promoter locus were constructed by in vitro mutagenesis, and their response to guanosine-5'-diphosphate-3'-diphosphate (ppGpp) was examined in a cell-free transcription system consisting of purified RNA polymerase holoenzyme . The nucleotide sequence (GpCpGpC) from positions -7 to -4 (designating the initiation site of mRNA as position +1) is responsible for the selective inhibition by ppGpp of tufB transcription . Point mutations were then constructed in which each one of the above four nucleotides was replaced by an A or T residue and tested for their response to ppGpp in the in vitro transcription system . The results indicated that the alteration of any nucleotide in the GpCpGpC sequence leads to the loss of the stringent response. EMBO J, 1985 Apr, 4(4), 1019 - 24 Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18? Christiansen J, Douthwaite SR, Christensen A, Garrett RA. Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18 . It has been proposed as a recognition site for protein L18 . We have investigated further the structural importance of this nucleotide by deleting it . The 5S RNA gene of the rrnB operon of E . coli was subjected to primer-directed mutagenesis . To produce the deletion it was necessary to use simultaneously the mutagenic dodecamer dCGGCGCACGGCG and the universal M13 primer dCCCAGTCACGACGTT, and to employ forced annealing conditions . The mutated gene was expressed in an overproducing plasmid derived from pKK3535 . Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA . We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18. Am J Vet Res, 1985 Apr, 46(4), 909 - 12 Nucleotide sequences of the genes for Escherichia coli heat-stable enterotoxin I of bovine, avian, and porcine origins; Sekizaki T et al.; The nucleotide sequences of cloned Escherichia coli heat-stable toxin 1 (STI) genes isolated from bovine, avian, and porcine origins were determined . They were found to be almost identical to that of Tn1681 . The nucleotide sequences were completely preserved in bovine and avian genes, whereas the porcine gene had different sequences at 3 positions in the external region of STI structural genes, as compared with Tn1681 . The amino acid sequences of the STI genes of the 3 animal origins corresponded to STIa, which had initially been found in a bovine strain. Genetika, 1985 Apr, 21(4), 541 - 7 {Organization of the genes responsible for colicin Ib synthesis and immunity to it in plasmid ColIb-P9}; Pshennikova ES et al.; To study the localization and expression of the ColIb-P9 plasmid genes responsible for colicin Ib synthesis and immunity to it, we isolated a series of Tn5 insertion mutants of recombinant plasmid pIV41 containing the colicin Ib gene in EcoRI fragment of ColIb-P9 (2.7 kb) and the deletion plasmid carrying only a part of the colicin gene . The direction of colicin Ib gene transcription was determined by the analysis of the polypeptides synthesized in minicells carrying the mutant plasmids . The pIV41 plasmid containing cells have been shown to be resistant to both colicin Ib and Ia activities . This type of resistance is usually associated with chromosomal mutations resulting in loss of cell receptors for colicin Ib adsorption . Apparently, the EcoRI fragment of ColIb-P9 studied contains no gene responsible for immunity to colicin . It has been shown that this gene is a portion of SalI fragment (22 kb) of the ColIb-P9, the fragment also carrying the gene which mediates synthesis of colicin Ib. Biochem J, 1985 Apr 1, 227(1), 287 - 97 The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype; Wright JK et al.; The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides {Wilson, Kusch & Kashket (1970) Biochem . Biophys . Res . Commun . 40, 1409-1414} . Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier . Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane . The pH-dependence of galactoside transport was compared in the mutant and the parent . At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose . At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant . The mutant also is unable to grow on melibiose at pH 5.5 . However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent . These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype . At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory . Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport . Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant . The reorientation of the unloaded carrier is slower in the mutant . However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose . These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2252 - 6 The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron; Vocke C et al.; The plasmid-encoded replication initiator protein of pSC101 specifically repressed initiation of transcription of its own cistron from its natural promoter . Addition of the purified initiator had little or no visible effect on transcription initiated from a heterologous promoter . DNase protection experiments revealed that the RNA polymerase recognition sequence was overlapped by the initiator protein recognition sequences, which are vicinal to the replication origin . Using the labeled promoter sequence, we have performed competitive DNase protection experiments in two ways: by adding RNA polymerase and initiator protein simultaneously or by sequentially adding first RNA polymerase and then initiator protein to the DNase reaction mixture . The RNA polymerase protection pattern was recessive to that of the initiator regardless of whether the two proteins were added simultaneously or sequentially . This observation suggests that the mechanism of autoregulation is due to competition of the two proteins for the sequences in and around the promoter region . Furthermore, the sequential addition experiments raise the possibility of displacement of RNA polymerase from the promoter by the initiator protein. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2244 - 6 The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene; Nyunoya H et al.; A near full-length cDNA copy of rat carbamoyl-phosphate synthetase I (EC 6.3.4.16) mRNA has been cloned . The cDNA insert in the recombinant plasmid pHN234 is 5.3 kilobases long . Analysis of the sequence coding for carbamoyl-phosphate synthetase I indicates that the gene has arisen from a fusion of two ancestral genes: one homologous to Escherichia coli carA, coding for a glutaminase subunit, and the second homologous to the carB gene that codes for the synthetase subunit . A short amino acid sequence previously proposed to be part of the active site involved in glutamine amide nitrogen transfer in the E . coli and yeast carbamoyl-phosphate synthetases (EC 6.3.5.5) is also present in the rat enzyme . In the mammalian enzyme, however, the glutaminase domain lacks a cysteine residue previously shown to interact with glutamine . The cysteine is replaced by a serine residue . This substitution could, in part, account for the inability of mammalian carbamoyl-phosphate synthetase I to catalyze the hydrolysis of glutamine to glutamic acid and ammonia. Mol Cell Biol, 1985 Apr, 5(4), 768 - 79 Two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides; Capon DJ et al.; The classical human interferon-alpha (HuIFN-alpha) gene family is estimated to consist of 15 or more nonallelic members which encode proteins sharing greater than 77% amino acid sequence homology . Low-stringency hybridization with a HuIFN-alpha cDNA probe permitted the isolation of two distinct classes of bovine IFN-alpha genes . The first subfamily (class I) is more closely related to the known HuIFN-alpha genes than to the second subfamily (class II) of bovine IFN-alpha genes . Extensive analysis of the human genome has revealed a HuIFN-alpha gene subfamily corresponding to the class II bovine IFN-alpha genes . The class I human and bovine IFN-alpha genes encode mature IFN polypeptides of 165 to 166 amino acids, whereas the class II IFN-alpha genes encode 172 amino acid proteins . Expression in Escherichia coli of members of both gene subfamilies results in polypeptides having potent antiviral activity . In contrast to previous studies which found no evidence of class II IFN-alpha protein or mRNA expression, we demonstrate that the class I and class II IFN-alpha genes are coordinately induced in response to viral infection. Mol Cell Biol, 1985 Apr, 5(4), 714 - 20 Homologous recombination catalyzed by human cell extracts; Kucherlapati RS et al.; Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells . We were able to obtain Neor colonies at a frequency of 2 X 10(-3) . This frequency was 100 to 1,000 times higher than that obtained with no extracts . The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies . Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination . On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro. J Bacteriol, 1985 Apr, 162(1), 413 - 9 Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187; Alatossava T et al.; Escherichia coli B cells were sensitized to ionophore A23187 by polymyxin B nonapeptide, and the induced magnesium and potassium ion fluxes were studied . Combined ionophore treatment permeabilized the cytoplasmic membrane of E . coli in an ion-specific manner and allowed the manipulation of intracellular Mg2+ content from the outside . A23187-induced Mg2+ efflux or influx was dependent on the free Mg2+ concentration gradient between the outside and inside of the cytoplasmic membrane and on the pH gradient . Most of the intracellular Mg2+ was bound, whereas only 1 to 2 mM was free in solution in the cellular sap. J Bacteriol, 1985 Apr, 162(1), 280 - 5 Cloning of the Escherichia coli recJ chromosomal region and identification of its encoded proteins; Lovett ST et al.; A 9.6-kilobase BamHI-SalI fragment carrying recJ+ was cloned into vector pBR322 . Deletion and transposon mutagenesis were used to map the recJ gene on this fragment . The maxicell protein-labeling technique was used to correlate a functional recJ gene with the presence of a polypeptide of 53,000 apparent molecular weight . Two additional genes, one encoding two proteins of 26,000 and 25,000 Mr and the other encoding a 31,000-Mr protein, were mapped on a 3.7-kilobase HindIII-SalI subfragment with recJ . Functions for these adjacent genes are not known; however, insertion mutations in these genes lessen the expression of the putative recJ protein detected in maxicells . A 9.6-kilobase BamHI-SalI fragment carrying the temperature-sensitive mutation recJ147 was also cloned and used for complementation studies to identify other recJ mutations. J Bacteriol, 1985 Apr, 162(1), 271 - 5 Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences; Gayda RC et al.; The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein . The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined . The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA . The DNA and protein sequences established that the mature protein is not processed from a precursor form . No sequence corresponding to an SOS box was found in the 5' sequence of DNA . There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding . The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene . A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences. J Bacteriol, 1985 Apr, 162(1), 256 - 62 Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr' prophage; Highton PJ et al.; Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent . The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2 . Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein . When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene . Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene . These results were supported by blot hybridization analysis of the E . coli chromosome with probes derived from the lc gene of phage PA-2 . A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence . We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E . coli K-12 map and that this gene can be activated by loss of an insertion element. J Bacteriol, 1985 Apr, 162(1), 155 - 61 Cold sensitivity induced by overproduction of UmuDC in Escherichia coli; Marsh L et al.; The UmuD and UmuC proteins of Escherichia coli are essential for mutagenesis by UV and most chemicals . Their mode of action is presently unknown . Strains which lack the LexA repressor {lexA(Def)} and contain a pBR322-derived plasmid carrying the umuDC operon overexpress UmuD and UmuC and become cold sensitive (growth at 42 degrees C but not at 30 degrees C) . Deletion mapping showed that the umuDC locus on the plasmid is responsible for conferring cold sensitivity . The conditional lethality appeared due to a rapid and reversible inhibition of DNA synthesis at the nonpermissive temperature . Cold sensitivity was enhanced by the increase of NaCl in the medium to 1% and eliminated by 4% ethanol in the medium . Cold sensitivity was partially suppressed by the lon-100 mutation and completely suppressed by the htpR165 mutation. Eur J Biochem, 1985 Apr 1, 148(1), 183 - 8 Chemical modifications of the Na+-H+ antiport in Escherichia coli membrane vesicles; Damiano E et al.; The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles . The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components . Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine . The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport . In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained. Eur J Biochem, 1985 Apr 1, 148(1), 113 - 8 A high-affinity site for glutathione in the cytoplasm of Escherichia coli and its possible role in potassium retention; Meury J et al.; Glutathione-deficient mutants of Escherichia coli, unlike the wild type, exhibit a fast leak and a fast turnover of K+ at steady state . In a medium with low K+ the growth rate is very slow when unsupplemented with glutathione; when supplemented with glutathione at a concentration as low as 0.1 microM the growth rate is similar to that of the wild type . The ability of glutathione to restore a wild-type growth rate can be accounted for by an immediate reduction of the K+ leak . Two analogs of glutathione also reduce the K+ leak: gamma-glutamylaminobutyrylglycine (ophthalmic acid) and alpha gamma-glutamylcystinylbisglycine . Glutathione binds with high affinity (Kd 50 nM) to a cytoplasmic protein; ophthalmic acid and alpha gamma-glutamylcystinylbisglycine compete with glutathione for the binding site (Ki 0.1 microM and 1 microM), thereby ruling out the possibility that the thiol is involved both in reducing the K+ leak and in binding to the high affinity binding site . For each of the three peptides the Kd for binding is similar to the minimal concentration that achieves the maximal reduction of the K+ leak . The results suggest that the binding of glutathione should play a role in the retention of K+ in E . coli. J Virol, 1985 Apr, 54(1), 21 - 9 Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells; Rea TJ et al.; RNA from pseudorabies virus (PRV)-infected cells was translated in a reticulocyte lysate with and without the addition of dog pancreas microsomes . Upon addition of the microsomes to the translation reaction, an additional prominent protein product was observed that was not present when microsomes were omitted . The gene coding for this processed protein and its lower-molecular-weight precursor was mapped within the small unique region of the genome by hybridization of mRNA to cloned fragments of PRV DNA and translation of the selected mRNAs . A fragment of the coding region of this gene was inserted into an open reading frame cloning vector to express part of this gene as a hybrid protein in Escherichia coli . This hybrid protein was injected into mice to raise an antiserum which was found to precipitate the glycoprotein which accumulates in the medium of PRV-infected cells . This allows us to conclude that the gene for the "excreted" glycoprotein (gX) maps to the small unique region of the genome, and that the precursor of this glycoprotein is readily processed by dog pancreas microsomes . The region of the PRV genome which codes for this glycoprotein was sequenced and found to include an open reading frame coding for 498 amino acids, flanked by sequences which contain features common to eucaryotic promoters and polyadenylation signals . The predicted protein sequence includes a hydrophobic sequence at the N-terminus which could be a signal sequence, and a hydrophobic sequence followed by a hydrophilic sequence at the C-terminus. J Virol, 1985 Apr, 54(1), 114 - 22 The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase; Prywes R et al.; Only 1.2 kilobases (kb) at the 5' end of the 3.9-kb v-abl sequence in Abelson murine leukemia virus is required for fibroblast transformation . A precise delineation of this minimum transforming region was made by using small 5' or 3' deletions . Insertions of four amino acids, generated by putting synthetic DNA linkers into various restriction enzyme cleavage sites, abolished transforming activity, indicating that much of the internal sequence of the minimum transforming region plays a critical role in the transformation process . This 5' 1.2 kb of v-abl encodes protein-tyrosine kinase activity when expressed in Escherichia coli . Each of the mutations which caused a loss of transformation activity also resulted in a loss of protein-tyrosine kinase activity when expressed in E . coli . The minimum transforming region of v-abl contains amino acid homology to other protein-tyrosine kinase oncogenes, and a comparison with these oncogenes is presented. Vet Microbiol, 1985 Apr, 10(3), 241 - 57 Molecular analysis of the virulence determinants of enterotoxigenic Escherichia coli isolated from domestic animals: applications for vaccine development; Dougan G et al.; Enterotoxigenic strains of Escherichia coli are an important cause of diarrhoeal disease in young farm animals . Several virulence determinants have been shown to play a major role in the pathogenicity of these strains . The molecular structure of some of these determinants including adhesion fimbriae, heat-labile toxins and heat-stable toxins have been elucidated . This knowledge has made possible the development of novel vaccines effective against enterotoxigenic strains . In this short review, the structure of these virulence factors will be described and the implications for the development of future vaccines will be discussed. J Clin Pathol, 1985 Apr, 38(4), 438 - 41 Verotoxin and neuraminidase induced platelet aggregating activity in plasma: their possible role in the pathogenesis of the haemolytic uraemic syndrome; Rose PE et al.; Certain strains of Escherichia coli producing verotoxin have been isolated in the stools of patients with the haemolytic uraemic syndrome . A platelet aggregating activity has been found in normal plasma after incubation with verotoxin at 37 degrees C for 24 h . This activity, unlike neuraminidase, has an effect that is independent of changing factor VIII related antigen, but requires the IIA and IIIB platelet surface glycoprotein (deficient in thrombasthenia) to mediate its effect . Prostacyclin totally inhibited this effect, but other antiplatelet drugs and heparin were without inhibitory effects. Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 1979 - 83 Expression of glnA in Escherichia coli is regulated at tandem promoters; Reitzer LJ et al.; We have determined that the glnA gene of the complex glnALG operon of Escherichia coli is transcribed from tandem promoters . Expression from the upstream promoter, glnAp1, requires the catabolite activating protein, is repressed by nitrogen regulator I (NRI), the product of glnG, and produces a transcript with an untranslated leader of 187 nucleotides . Expression from the downstream promoter, glnAp2, requires NRI as well as the glnF product; full expression also requires growth in a nitrogen-limited environment . The downstream transcript has an untranslated leader of 73 nucleotides . We also provide evidence that the function of the glnL product is to mediate the interconversion of NRI between a form capable of activating glnAp2 and an inactive form in response to changes in the intracellular concentration of ammonia . The function of the two minor promoters of the glnALG operon, glnAp1 and glnLp, is to maintain the products of glnA, glutamine synthetase, an essential enzyme, and of glnG, NRI, an activator of nitrogen-controlled genes, during carbon-limited growth. J Bacteriol, 1985 Apr, 162(1), 454 - 7 Nucleotide sequence of pilA, the gene encoding the structural component of type 1 pili in Escherichia coli; Orndorff PE et al.; The pilA gene of Escherichia coli J96 encoding pilin, the structural component of type 1 pili, was sequenced and found to specify a polypeptide 159 amino acids long preceded by a 23-amino-acid signal peptide . As determined from the DNA sequence, the mature peptide lacked tryptophane and methionine, two amino acids previously shown to be lacking in type 1 pili from E . coli . Also, the amino-terminal sequence of amino acids inferred from the DNA sequence corresponded to earlier 20-amino-acid amino-terminal sequences determined by protein sequencing . In addition, piliation was abolished after a mutation was introduced into the pilA coding region in vitro . A possible site for initiation of transcription and a possible site encoding translation initiation were suggested 85 and 7 base pairs, respectively, from the pilA start codon . There appeared to be scant DNA sequence homology and scant amino acid sequence homology between type 1 pilin and other pilin species isolated from uropathogenic and enteropathogenic E . coli. J Bacteriol, 1985 Apr, 162(1), 420 - 6 Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli; Jans DA et al.; A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo . The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome . The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation . Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable . It was concluded that the F0 sector was assembled . Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131 . Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp) . Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages. J Bacteriol, 1985 Apr, 162(1), 391 - 7 Release of cell wall peptides into culture medium by exponentially growing Escherichia coli; Goodell EW et al.; Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala) . From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation. Infect Immun, 1985 Apr, 48(1), 211 - 8 Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites; Feingold C et al.; Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity) . A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed . Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region . The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid . Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity . Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups . The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds . The materials from both peaks had enterotoxic activity in intestinal ligated loops . The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca . 30,000 on sodium dodecyl sulfate . Amino acid analysis revealed that the protein was very poor in sulfur amino acids . The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains. DNA, 1985 Apr, 4(2), 127 - 37 The sequence of 16S rRNA from Mycoplasma strain PG50; Frydenberg J et al.; The DNA sequence of one of the 16S rRNA genes (cistron rrnA) of Mycoplasma strain PG50 was determined . It is 1523 bp long and has about 70% homology to the sequence of Escherichia coli 16S rRNA (rrnB) . The G + C content of the sequence is 48% compared with 56% G + C in the E . coli sequence . The secondary structure is formed and it is determined that most of the differences between the two sequences are seen in stems while loops in the secondary structure are conserved . A detailed description of differences and similarities to known sequences and rRNA oligonucleotide catalogues is given, and this information is used to discuss functional properties and phylogenetic relations of mycoplasma 16S rRNA. J Gen Microbiol, 1985 Apr, 131 ( Pt 4), 945 - 9 Interactions between mutations affecting ribosome synthesis in Escherichia coli; Butler PD et al.; RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA . The ribosome mutation did not by itself lead to relaxedness . The relaxed mutation could be expressed in organisms that contained the ribosome mutation. Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 1911 - 5 Escherichia coli transcription termination factor rho has a two-domain structure in its activated form; Bear DG et al.; Limited tryptic digestion of Escherichia coli transcription termination factor rho {an RNA-dependent nucleoside triphosphatase (NTPase)} yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP . The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa) . Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283 . These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage . The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity . ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis . The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-{gamma-thio}triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand . These findings are discussed within the context of recent models of rho-dependent transcription termination. Mutat Res, 1985 Apr, 142(4), 159 - 62 Influence of the recB21 mutation of Escherichia coli K12 on prophage lambda induction; Simic D et al.; The inactivation kinetics of the lambda repressor following bleomycin (BM), UV-irradiation and nalidixic acid (NAL) treatments were studied in the recB21 mutant of E . coli K12 . The results showed essentially normal induction by UV-irradiation, delayed induction by BM and no induction by NAL . The results were compared with inactivation kinetics in lexA1 and recF143 mutants. J Bacteriol, 1985 Apr, 162(1), 85 - 91 Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin; Gabay JE et al.; We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin . To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield . The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent . The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography . The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C . To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure . Like the Escherichia coli porins, the MOMP was peptidoglycan associated . The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration . To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes . The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens . The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating . These findings demonstrate that the MOMP is a porin with properties similar to those of E . coli porins. J Bacteriol, 1985 Apr, 162(1), 458 - 60 Effect of DNA gyrase inactivation on RNA synthesis in Escherichia coli; Wahle E et al.; The average chain growth rates of rRNA and of total RNA were not affected by a thermal inactivation of DNA gyrase in a temperature-sensitive gyrB mutant of Escherichia coli . The fact that total RNA synthesis decreased under these conditions suggests that transcription is primarily affected at the step of chain initiation . The fraction of rRNA in total pulse-labeled RNA was not altered by inactivation of the enzyme, indicating that the latter is not required to actively maintain a high rate of synthesis of this RNA species. J Bacteriol, 1985 Apr, 162(1), 117 - 23 New gene in Escherichia coli K-12 (drpA): does its product play a role in RNA synthesis? Lech KF, Lee CH, Isberg RR, Syvanen M. The mutation drpA1 defines a new gene in Escherichia coli K-12 that maps at about 5.2 min . This mutation was obtained after enriching a population of cells for temperature sensitive dna mutations with the {3H}thymidine "suicide" technique followed by screening for mutants defective in transposon Tn5 precise excision . When growing cells carrying the drpA1 allele were shifted to the nonpermissive temperature, we showed that DNA, RNA, and protein syntheses shut off quickly, with the cessation of RNA synthesis occurring first . A recombinant plasmid between pBR322 and an HindIII fragment from wild-type E . coli restores the growth defect in drpA1 mutants . Using transposon Tn5 mutagenesis of this plasmid, we have been able to correlate the presence of a 68-kilodalton protein, as observed with the maxicell technique, with the ability of this plasmid to restore growth to drpA1 mutants. Eur J Biochem, 1985 Apr 1, 148(1), 1 - 5 Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities; Galanos C et al.; The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E . coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity . It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests. J Immunol, 1985 Apr, 134(4), 2414 - 8 Interleukin 2-mediated induction of lytic activity in a cloned murine CTL line; Howe RC et al.; We have analyzed the ability of interleukin 2 (IL 2) to induce lytic activity within a cloned murine H-2Dd-specific CTL line . Weakly lytic CTL harvested 6 to 7 days after previous stimulation with irradiated DBA/2J spleen cells and conditioned medium from secondary MLC (MLC SN) could be reactivated to high antigen-specific lytic activity with highly purified gibbon IL 2 or E . coli-produced human recombinant DNA IL 2 . Dose-response curves with IL 2 and MLC SN suggest that IL 2 may be the principal detectable activity in MLC SN that is active on these CTL . Doses of IL 2 or MLC SN that were saturating for the induction of lytic activity were suboptimal for the expression of DNA synthesis measured by 3HTdR incorporation . This is consistent with a mechanism in which different threshold IL 2 concentrations are required to induce these two biologic responses . Finally, we show that IFN-gamma has little effect on the expression of lytic activity either alone or in combination with IL 2 in this bioassay. Avian Dis, 1985 Apr-Jun, 29(2), 540 - 5 Immunogenicity of an oil-emulsified Escherichia coli bacterin against heterologous challenge; Gyimah JE et al.; Immunogenicity of an oil-emulsified Escherichia coli bacterin against heterologous challenge was investigated . In Expts . 1 and 2, chickens were vaccinated with E . coli serotype O1 bacterin and challenged with E . coli serotype O2 (Expt . 1) and O78 (Expt . 2) . Positive control chickens were not vaccinated but challenged with E . coli serotype O2 or O78; unvaccinated unchallenged chickens served as negative controls . When challenged with E . coli serotype O2, unvaccinated chickens showed a higher morbidity than vaccinated chickens . There was no mortality in either group . Although average gross lesion scores were generally higher in the unvaccinated chickens, they were not significantly different (P greater than or equal to 0.05) from those in the vaccinated chickens . In Expt . 2, morbidity was slightly higher in the unvaccinated challenged chickens . No mortality occurred in either group . There was no significant difference (P greater than or equal to 0.05) between vaccinated and unvaccinated chickens in average gross lesion scores . In general, E . coli recovery was higher in the unvaccinated challenged chickens, being highest in the air sacs followed by the liver, heart blood, and pericardial sacs . There was no morbidity, mortality, or gross lesions in the unvaccinated unchallenged chickens . No E . coli was recovered from the tissues cultured . The results of these laboratory trials revealed that an oilemulsified monovalent E . coli bacterin did not protect chickens against other E . coli serotypes associated with colibacillosis. Can J Comp Med, 1985 Apr, 49(2), 179 - 85 Calcium mediation of the pig jejunal secretory response; Forsyth GW et al.; The involvement of Ca++ ions as secretory mediators in pig jejunal epithelia has been investigated with an in vitro system . Omission of Ca++ from the Ringer-HCO3 bathing media on both sides of the tissue had minor effects on the basal electrical activity of pig jejunal mucosa . There were only slight decreases in transepithelial potential difference and increases in conductance with Ca++ free media . Low EGTA concentrations which reversibly blocked potential difference responses to secretory agents also had minimal effects on basal electrical activity . The in vitro secretory responses to A23187, to theophylline, and to Escherichia coli heat-stable enterotoxin were all eliminated by Ca++ depletion and restored by replacing normal Ca++ concentrations in the bathing media . Dantrolene prevented the secretory response but not the potential difference increases caused by heat-stable enterotoxin and A23187, suggesting that intracellular Ca++ stores may be reservoirs of secretory signal agent . Verapamil only blocked the secretory response to heat-stable enterotoxin . Chlorpromazine had negligible effects on basal conditions, but totally blocked both the secretory response and the Ca++-dependent effects of A23187 and heat-stable enterotoxin on potential difference . The response to theophylline was only partially inhibited by chlorpromazine, implying some involvement of both cAMP and Ca++ as secretory signals for theophylline . Cytoplasmic Ca++ concentrations appear to be at least as important as cyclic nucleotides in regulating the secretory effects of pig jejunum. Clin Exp Immunol, 1985 Apr, 60(1), 196 - 202 Inverse relationship between interferon production by mononuclear leucocytes and oxidative metabolism of neutrophil granulocytes in infection prone children; Bondestam M et al.; The ability of neutrophil granulocytes (PMN) from 15 infection prone children to produce luminol enhanced chemiluminescence (CL) was disturbed to varying extents in eight patients, two of whom were diagnosed as having chronic granulomatous disease (CGD) . The ability of peripheral blood mononuclear leucocytes (PBL) to produce interferons (IFN) was normal, as tested with the inducers Sendai virus, E . coli, concanavalin A and L . culinaris lectin . However, the IFN response to the inducer S . aureus Cowan I (SACoI) was decreased in patients with normal CL and tended to be increased in those with decreased CL . There was a significant inverse relation between the CL of PMN and the SACoI-induced IFN responses by PBL of the patients . A regulatory effect of products of oxidative metabolism on SACoI-induced IFN production is therefore suggested . Patients, including those with CGD and controls showed similar basal and in vitro IFN enhanced natural killer (NK) activity of PBL against K-562 erythroleukaemia cells. Bioorg Khim, 1985 Apr, 11(4), 492 - 8 {The role of sulfhydryl groups in the functioning of DNA-dependent RNA-polymerase}; Chertov OIu et al.; Sulfhydryl groups of Escherichia coli DNA-dependent RNA polymerase were chemically modified with alkylating and mercuric-containing compounds . Iodoacetic acid and iodoacetamide were shown not to affect the enzymatic activity, whereas N-ethylmaleimide and mercuric-containing compounds completely inhibit the RNA synthesis . RNA polymerase modified with mercuric ions looses the ability of binding with promoter--containing DNA fragments . Moreover, mercuric ions inhibit the RNA elongation stage . Suggestion is made the Cys residues of RNA polymerase play a key role in double-stranded DNA unwinding . It is shown that SH-groups of beta- and beta'-subunits participate in the binding with double-stranded fragments of DNA. Scand J Immunol, 1985 Apr, 21(4), 337 - 43 Hapten-specific unresponsiveness in mice . III . Study of the B-cell functions of tolerant mice; Huchet R et al.; The features of B-cell tolerance induced in mice with the chemically reactive hapten 2,4,6-trinitrobenzenesulphonic acid were investigated after various antigenic challenges . A complete abolition of the IgG response was observed after challenge with trinitrophenol (TNP)-coupled haemocyanin (TNP-KLH) associated or not with Escherichia coli lipopolysaccharide . Carrier immunization with KLH (3 micrograms) followed by challenge with TNP-KLH led to a normal IgG anti-TNP response . However, tolerant mice given 3 micrograms TNP-KLH (instead of KLH) and a repeat injection of TNP-KLH exhibited a depressed IgG anti-TNP response . These results are discussed within the framework of tolerance induction in adult mature B cells. Biochim Biophys Acta, 1985 Mar 29, 839(1), 32 - 9 Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase; West TP et al.; Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated . The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes . The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution . The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the holoenzyme . The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins . Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity . Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration . A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented . This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures. Science, 1985 Mar 29, 227(4694), 1593 - 7 Isolation of the gene for a glycophorin-binding protein implicated in erythrocyte invasion by a malaria parasite; Ravetch JV et al.; Plasmodium falciparum, the most lethal of the malarial parasites that infect humans, undergoes three cycles of development in its vertebrate host and elicits stage-specific immune responses . This stage specificity of the immune response has made it difficult to isolate antigens that would be useful in developing a vaccine against malaria . A complementary DNA clone for a glycophorin-binding protein of Plasmodium falciparum merozoites has been isolated and characterized . The protein interacts with glycophorin, the erythrocyte receptor, during invasion of the host cell by the parasite . Antigenic determinants of this protein expressed in Escherichia coli have been used to produce antibodies to a glycophorin-binding protein . The antibodies show schizont-specific immunofluorescence and react with the merozoite protein . The primary sequence of these determinants reveals a 150-nucleotide tandem-repeating sequence coding for a 50-amino-acid repeat . The characterization of the Plasmodium falciparum glycophorin-binding protein represents one approach toward designing serologic agents to block the parasite's development in the vertebrate host. Biochem Biophys Res Commun, 1985 Mar 29, 127(3), 799 - 808 Platelet activating factor (PAF) involvement in endotoxin-induced hypotension in rats . Studies with PAF-receptor antagonist kadsurenone; Doebber TW et al.; Evidence from three types of experiments indicates that platelet activating factor (PAF)1 is an important mediator of endotoxin-induced hypotension in rats . a) Endotoxin infusion stimulates the time-dependent appearance of PAF in the blood . b) PAF infusion results immediately (less than 30 sec) in hypotension while endotoxin-induced hypotension takes 3-5 min to occur, allowing time for PAF production . c) Infusion of the specific PAF-receptor antagonist kadsurenone (2.2 mumole/kg bolus, 0.9 mumoles/min/kg continuous infusion), which inhibits PAF-induced hypotension by 67%, causes a 67% reversal of endotoxin-elicited hypotension . An additional finding of this study is that rats respond hypotensively to each of a series of low-dose PAF infusions but only to the first low-dose endotoxin infusion . These endotoxin-refractory rats do respond to subsequent PAF infusions. Nucleic Acids Res, 1985 Mar 25, 13(6), 2017 - 34 Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library; Hallewell RA et al.; The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported . The tacI promoter has been used to direct the synthesis in E . coli of this SOD which is soluble, stable, and of normal specific activity . The N-terminal methionine is removed from this protein . A construction with a ribosome binding site identical to that of the lacz gene 5' of the initiator methionine codon, resulted in low levels of SOD . An in vitro mutagenesis procedure was used to randomize the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids . Analysis of a sample of 500 clones showed that ca . 25 clones synthesised 5% or more of soluble cell protein as SOD . The nucleotide sequences of high level expressors showed a predominance of A and T residues in the variable positions 5' of the initiator methionine codon . An SOD mutant (ala4----gln) was discovered during the sequencing and shown to lack dismutation activity . Secondary structure predictions for the 5' regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to 16s rRNA is base paired in a stem structure. FEBS Lett, 1985 Mar 25, 182(2), 253 - 6 Protein S4 is near the elongation factor G binding site in the ribosome; Tejedor F et al.; Using a mild iodination method for protein radioactive labeling, it has been shown that elongation factor G, when bound to the ribosome as EFG-GDP-fusidic acid complex, protects protein S4 from labeling . The results indicate that protein S4 is probably near the ribosomal EFG binding site. J Biol Chem, 1985 Mar 25, 260(6), 3594 - 603 Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes . Modulation by NaCl concentration; Lohman TM et al.; The binding properties of the Escherichia coli encoded single strand binding protein (SSB) to a variety of synthetic homopolynucleotides, as well as to single stranded M13 DNA, have been examined as a function of the NaCl concentration (25.0 degrees C, pH 8.1) . Quenching of the intrinsic tryptophan fluorescence of the SSB protein by the nucleic acid is used to monitor binding . We find that the site size (n) for binding of SSB to all single stranded nucleic acids is quite dependent on the NaCl concentration . For SSB-poly(dT), n = 33 +/- 3 nucleotides/tetramer below 10 mM NaCl and 65 +/- 5 nucleotides/tetramer above 0.20 M NaCl (up to 5 M) . Between 10 mM and 0.2 M NaCl, the apparent site size increases continuously with {NaCl} . The extent of quenching of the bound SSB fluorescence by poly(dT) also displays two-state behavior, 51 +/- 3% quenching below 10 mM NaCl and 83 +/- 3% quenching at high {NaCl} (greater than 01.-0.2 M NaCl), which correlates with the observed changes in the occluded site size . On the basis of these observations as well as the data of Krauss et al . (Krauss, G., Sindermann, H., Schomburg, U., and Maass, G . (1981) Biochemistry 20, 5346-5352) and Chrysogelos and Griffith (Chrysogelos, S., and Griffith, J . (1982) Proc . Natl . Acad . Sci . U . S . A . 79,5803-5807) we propose a model in which E . coli SSB binds to single stranded nucleic acids in two binding modes, a low salt mode (n = 33 +/- 3), referred to as (SSB)33, in which the nucleic acid interacts with only two protomers of the tetramer, and one at higher {NaCl}, n = 65 +/- 5, (SSB)65, in which the nucleic acid interacts with all 4 protomers of the tetramer . At intermediate NaCl concentrations a mixture of these two binding modes exists which explains the variable site sizes and other apparent discrepancies previously reported for SSB binding . The transition between the two binding modes is reversible, although the kinetics are slow, and it is modulated by NaCl concentrations within the physiological range . We suggest that SSB may utilize both binding modes in its range of functions (replication, recombination, repair) and that in vivo changes in the ionic media may play a role in regulating some of these processes. J Biol Chem, 1985 Mar 25, 260(6), 3529 - 38 Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter; Mulligan ME et al.; We describe the characterization in vitro of three hybrid trp-lacUV5 (TAC) promoters, which have perfect homology to the -10 and -35 Escherichia coli promoter consensus hexamer sequences, but which differ in the distance between the -10 and -35 regions . The three promoters, TAC16, TAC17, and TAC18 have spacings of 16, 17, and 18 base pairs, respectively . We have measured KB and k2, the constants that describe the formation of open complexes, on these three promoters . We have also measured their relative strengths on supercoiled plasmid DNA . Our results show that these are the strongest promoters that have been characterized in vitro so far and confirm the hypothesis that the consensus promoter sequence is "best." We find the TAC17 promoter (KBk2 = 8.4 X 10(7) M-1 s-1) to be stronger than either the TAC16 (KBk2 approximately 1.5 X 10(7) M-1 s-1) or TAC18 (KBk2 approximately 3.5 X 10(7) M-1 s-1) promoters, a result that is in agreement with other findings on the effect of spacer length . The choice of start point for transcription is affected by spacer length . Transcription from all the promoters was stimulated at moderate concentrations of salt (less than 150 mM) and persisted at high salt concentrations (300 mM). Nucleic Acids Res, 1985 Mar 25, 13(6), 1891 - 903 Molecular cloning and nucleotide sequence of rat lingual lipase cDNA; Docherty AJ et al.; Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme . These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E . coli expression vectors . An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained . Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues . The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme. Nucleic Acids Res, 1985 Mar 25, 13(6), 2063 - 74 Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W; Takagi JS et al.; The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322 . The nucleotide sequences of aspA and its flanking regions were determined . The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues . The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously . Potential promoter and terminator sequences for aspA were also found in the determined sequence. J Biol Chem, 1985 Mar 25, 260(6), 3697 - 702 A linear double-stranded RNA in Trichomonas vaginalis; Wang AL et al.; A "double-stranded" RNA was identified in the anaerobic, parasitic protozoan Trichomonas vaginalis . Electron microscopic evidence indicated linear double-stranded structure 1.5 micron in length, with no apparent hairpins or loops . Boiling in 30% dimethyl sulfoxide denatured it into single strands of 1.5 micron and shorter fragments . It consists of 23.4% G, 23.4% C, 23.0% A, and 30.3% U and melts at a transition temperature of 81.7 degrees C in 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 7-15% hyperchromicity . The 32P-labeled double-stranded RNA hybridized specifically with T . vaginalis DNA fragments in a single DNA band from EcoRI digest and two DNA bands from HindIII digest . Of the 33 different strains or isolates of T . vaginalis examined, all contained this double-stranded RNA . However, the only two metronidazole-resistant T . vaginalis strains examined thus far (IR78 and 85) contained no detectable double-stranded RNA, although the corresponding DNA sequence was present . DNA fragments of Escherichia coli and Giardia lamblia did not hybridize with the double-stranded RNA . But DNA fragments of a metronidazole-sensitive Tritrichomonas foetus hybridized specifically with the double-stranded RNA, even though this organism does not contain the double-stranded RNA itself. J Biol Chem, 1985 Mar 25, 260(6), 3652 - 7 Isolation and analysis of an Abelson murine leukemia virus-encoded tyrosine-specific kinase produced in Escherichia coli; Ferguson B et al.; A segment of the coding sequence of the Abelson murine leukemia virus transforming gene (v-abl) has been inserted into a plasmid vector that allows its efficient and regulated expression in Escherichia coli . The product of the v-abl-derived coding sequence, designated p60v-abl, accumulated to a level of approximately 10% of total E . coli protein . A procedure is described for the isolation of p60v-abl from E . coli that yields about 50 micrograms of p60v-abl/g wet weight of E . coli . p60v-abl was capable of autophosphorylation and phosphorylating certain E . coli proteins specifically at tyrosine residues . The E . coli-expressed p60v-abl specifically phosphorylated tyrosine residues on casein and angiotensin II . The Km and Vmax values for ATP, casein, and angiotensin II in the p60v-abl kinase reaction have been determined and compared to values reported for other tyrosine-specific kinases . The expression system and isolation procedure described here permit the preparation of functional p60v-abl in quantities sufficient for detailed physical and biochemical characterization and examination of its biological action(s). J Biol Chem, 1985 Mar 25, 260(6), 3350 - 4 Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase; Zalkin H et al.; An improved method was developed to align related protein sequences and search for homology . A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli . Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II . This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase . Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins . Two rules are formulated to explain trpG and trpG-related fusions . (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion . (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon . These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively . Alignment searches of GMP synthetase with two other enzymes that bind GMP, E . coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site. J Biol Chem, 1985 Mar 25, 260(6), 3820 - 5 Operator mutations of the Escherichia coli aroF gene; Garner CC et al.; The Escherichia coli aroF and aroG genes encode the tyrosine-sensitive and the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, respectively, two of the three isoenzymes that control carbon flow through the shikimate pathway . Transcription of aroF and aroG is repressed by the tyrR gene product complexed to tyrosine or phenylalanine, respectively . Constitutive aroF mutants with lesions linked to aroF were isolated . The nucleotide sequences in the regulatory regions of aroF from six such mutants and from the parental wild type strain were determined . The mutations were found in two 18-base pair imperfect palindromes, called aroFo1 and aroFo2, which are located upstream of the aroF transcription start by 61 and 113 base pairs, respectively . Nuclease S1 mapping and analysis of in vitro run-off transcripts identified the 5'-end of the aroF transcript 51 base pairs upstream of the aroF translation start . The -35 region of the aroF promoter overlaps aroFo1 . The aroFo1 and aroFo2 sequences are homologous to a single 18-base pair DNA segment preceding the coding sequence of aroG. FEBS Lett, 1985 Mar 25, 182(2), 407 - 12 Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain; Mackow ER et al.; An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E . coli relaxed strain KL99 . This E . coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA . When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content {Mol . Gen . Genet . (1983) 192, 5-9} appeared to cause a reduction in new rRNA synthesis . Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively . This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation. J Biol Chem, 1985 Mar 25, 260(6), 3539 - 41 Spacing of the -10 and -35 regions in the tac promoter . Effect on its in vivo activity; Brosius J et al.; In the tac promoter (deBoer, H . A., Comstock, L . J., and Vasser, M . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 21-25) the spacing between the -35 and -10 consensus sequences is 16 base pairs . Between these two regions we inserted 1 or 2 base pairs to increase the distance to 17 base pairs (trc promoter) or 18 base pairs (tic promoter) . The activities of the three promoters were compared in vivo by fusion to the chloramphenicol acetyltransferase or to the Escherichia coli 4.5 S RNA gene . Both measurements gave consistent results . The trc and tic promoters are on average about 90% and 65% as active as the tac promoter, respectively. J Mol Biol, 1985 Mar 20, 182(2), 217 - 27 Colicin E3 and its immunity genes; Masaki H et al.; A DNA segment of plasmid ColE3-CA38 was cloned into pBR328 and its nucleotide sequence was determined . This segment contains the putative promoter-operator region, the structural genes of protein A (gene A) and protein B (gene B) of colicin E3, and a part of gene H . Just behind the promoter region, there is an inverted repeat structure of two 'SOS boxes', the specific binding site of the lexA protein . This suggests that the expression of colicin E3 is regulated directly by the lexA protein . Genes A and B face the same direction, with an intergenic space of nine nucleotides between them . ColE3-CA38 and ColE1-K30 are homologous in their promoter-operator regions, but hardly any homology was found in their structural genes . On the other hand, ColE3-CA38 is fairly homologous to CloDF13 throughout the regions sequenced, with some exceptions including putative receptor-binding regions . By deletion mapping of the immunity gene and recloning of gene B, it was shown genetically that protein B itself is the actual immunity substance of colicin E3 . It was also found that the expression of E3 immunity partially depends on the recA function . Thus, we propose two modes of expression of E3 immunity: in the uninduced state, only a slight amount of protein B is produced constitutively to protect the cell from being attacked by the exogenous colicin; and in the SOS-induced state, a large amount of protein B is produced to protect the protein synthesis system of the host cell from ribosome inactivation by endogenously produced colicin E3. J Mol Biol, 1985 Mar 20, 182(2), 241 - 8 The 5' ends of Escherichia coli lac mRNA; Cannistraro VJ et al.; We identified the predominant 5' ends of an mRNA in Escherichia coli to the exact nucleotides . There are four such ends of lac mRNA in fully induced cells . About 70% of the molecules have the reported major in vitro end, A-A-U-U-G (at +1), which is located 38 nucleotides before the A-U-G translation start . Another 15% start with A-U-U-G at +2, and about 8% start with A-U-U-A-G at -52 . A fourth class of molecules begin with either A-G, C-A-G, A-C-A-G, or a weak A-C-A-C-A-G (at +24), observed only once . The origins of this latter set (less than or equal to 10% of the total) are not known, but they could represent "ragged" ends of the mRNA when it is degraded to the beginning of the ribosome-protected region of the message . The A-U-U-A-G molecules are probably initiated from an upstream promoter whose position would coincide with the cAMP-CRP DNA binding site for the major promoter. J Mol Biol, 1985 Mar 20, 182(2), 205 - 16 A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro; Stark MJ et al.; We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene . The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript . Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation . Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme. J Immunol Methods, 1985 Mar 18, 77(2), 275 - 82 A two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies; Tanaka E et al.; Two monoclonal antibodies (nos . 6008 and 6016) were raised against human gamma interferon (IFN-gamma) derived from E . coli harboring the recombinant cDNA for IFN-gamma, and one (3710) against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues . The monoclonal antibody against the synthetic peptide (3710) reacted either with IFN-gamma or the synthetic peptide . One monoclonal anti-IFN-gamma (6008) did not react with the synthetic peptide, while the other (6016) showed a weak binding with the peptide . The binding of the monoclonal antibody against the synthetic peptide (3710) with IFN-gamma was not inhibited by 6008, but to a certain extent by 6016 . A 2-site '1-step' radioimmunoassay was developed in which 6008 was fixed on a solid-phase support, and the test sample together with radiolabeled 3710 was added for the binding with it . The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-gamma. J Chromatogr, 1985 Mar 15, 321(2), 385 - 91 Headspace gas chromatographic determination of beta-galactosidase activity using electron-capture detection; Koppen B et al.; A headspace gas chromatographic method for the determination of beta-galactosidase (E.C . 3.2.1.23) activity is described . The method, in which 2,2,2-trichloroethyl beta-D-galactopyranoside (beta-TCG) is used as substrate, involves determination of the liberated 2,2,2-trichloroethanol by gas chromatography with electron capture detection . The preparation of beta-TCG and of 2,2,2-trichloroethyl alpha-D-galactopyranoside is described . A Km = 0.80 mM was found for the enzymatic hydrolysis of beta-TCG employing beta-galactosidase from Escherichia coli . The assay has been applied to the quantitative determination of E . coli bacteria. Eur J Biochem, 1985 Mar 15, 147(3), 601 - 9 Structure of the fructose-containing K52 capsular polysaccharide of uropathogenic Escherichia coli O4:K52:H-; Hofmann P et al.; The chemical structure of the K52 antigenic capsular polysaccharide (K52 antigen) of Escherichia coli O4:K52:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation before and after graded acid hydrolysis and by oligosaccharide analysis . The polysaccharide consists of a backbone of alpha-galactose units interlinked between C1 and C3 by phosphodiester bridges . This poly(alpha-galactosyl-phosphate) is substituted at C2 of each galactose unit by beta-fructofuranose residues . About 80% of the galactose units are O-acetylated at C4 and about 10% of the fructose units are both O-acetylated and O-propionylated at C1 . The K52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units. Science, 1985 Mar 15, 227(4692), 1340 - 3 Heterogeneity of 5S RNA in fungal ribosomes; Selker EU et al.; Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions . A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha) . A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi . In addition, different 5S RNA's are present in Neurospora ribosomes . The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well. FEBS Lett, 1985 Mar 11, 182(1), 77 - 80 Interaction of bovine seminalplasmin with Escherichia coli RNA polymerase in the presence of rifampicin; Sitaram N et al.; The interaction of bovine seminalplasmin and rifampicin with E . coli RNA polymerase was studied using fluorescence spectroscopy . Both seminalplasmin and rifampicin are known to be the inhibitors for the initiation of RNA synthesis in E . coli . Rifampicin quenced the intrinsic fluorescence of RNA polymerase and seminalplasmin when excited at 280 nm . However, excess of seminalplasmin reversed the quenching of RNA polymerase fluorescence by rifampicin . Upon addition of rifampicin to the seminalplasmin-RNA polymerase complex, no change in fluorescence spectrum was observed . It appeared that although rifampicin could form complexes with RNA polymerase and seminalplasmin alone, no binding domain was available for rifampicin in the RNA polymerase-seminalplasmin complex . These observations are discussed in the light of the 'initiation site' of E . coli RNA polymerase. Nucleic Acids Res, 1985 Mar 11, 13(5), 1483 - 92 Stimulation of the UvrABC enzyme-catalyzed repair reactions by the UvrD protein (DNA helicase II); Kumura K et al.; An in vitro assay system was constructed using highly purified preparations of UvrA, UvrB, UvrC, UvrD proteins and DNA polymerase I, the objective being to analyse the role of UvrD protein in excision repair of UV-induced DNA damage . UvrABC enzyme-initiated repair synthesis was greatly enhanced by the addition of UvrD protein to the reaction mixture . Further analysis revealed that UvrD protein stimulated introduction of strand breaks in irradiated DNA by UvrABC enzyme but had no effect on the DNA polymerase I reaction . Thus, the site of action of UvrD protein is probably at the incision-excision step and not in later steps in excision repair. J Biol Chem, 1985 Mar 10, 260(5), 3050 - 7 Structure and assembly of the endoplasmic reticulum . The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes; Lewis MJ et al.; Monospecific rabbit antibodies have been prepared against ERp72, ERp99, and ERp60, major protein components of a detergent-solubilized extract of endoplasmic reticulum purified from mineral oil-induced plasmacytoma 315 tissue . When subcellular fractions of mineral oil-induced plasmacytoma 315 tissue were assayed by an immunoprecipitation procedure, all three endoplasmic reticulum proteins (ERps) were found to be enriched in the rough endoplasmic reticulum . In murine lymphoid cells, the three ERps represent two major structural classes of protein . Both ERp72 and ERp60 contain no endoglycosidase H-sensitive, N-linked oligosaccharides . On the other hand, ERp99 is glycoprotein containing, in all likelihood, one endoglycosidase H-sensitive oligosaccharide . Immunologically cross-reacting proteins of similar molecular weight have also been detected in other eukaryotic cell lines . The anti-ERp antibodies were used to quantitate the synthesis and accumulation of the three ERps in splenic lymphocytes cultured in the presence and absence of bacterial lipopolysaccharide (Escherichia coli serotype B5:055) (LPS) . In the presence of LPS, lymphocytes differentiate from resting cells into actively secreting cells . The synthesis of ERp72 and ERp99 increased 3- and 10-fold, respectively, in response to LPS . The synthesis of ERp60 does not change significantly . The turnover rates for these three proteins are similar in both control and LPS-treated lymphocytes . As a result, membranes isolated from LPS-treated cells are enriched in ERp72 and ERp99. J Biol Chem, 1985 Mar 10, 260(5), 3178 - 84 Characterization of DNA polymerase I*, a form of DNA polymerase I found in Escherichia coli expressing SOS functions; Lackey D et al.; DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions . Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative . Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants . Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S . Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels . Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides . Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable . Estimates of processivity give a value of approximately 6 for both forms of the enzyme. J Biol Chem, 1985 Mar 10, 260(5), 2875 - 83 Alkaline phosphatase . 31P NMR probes of the mechanism; Gettins P et al.; 31P NMR signals from substrates and products of alkaline phosphatase have been adapted to measure the rates and product ratios for the hydrolysis and phosphotransferase reactions from pH 6 to 10 . Below pH 8, glycerol is a poorer acceptor than H2O (glycerol phosphates:Pi = 0.5) . Tris is a more effective acceptor below pH 8, showing a maximum acceptor efficiency at pH 8 (Tris phosphate:Pi = 2) . Phosphotransferase efficiencies are in the order expected for the pKaS of the alcohol groups, Tris less than glycerol Cl, C3 less than glycerol C2 . Tris and glycerol induce chemical shifts in 113Cd(II) present at the A site but not the B or C sites of the metal triad present at each active center of Cd(II)6 alkaline phosphatase, suggesting that the alcoxides of the acceptors coordinate the A site metal and become the nucleophiles attacking the phosphoseryl residue (E-P) in the second step of the mechanism . The interaction is through the oxygen of Tris . The transferase activity of the amino alcohol shows a bell-shaped pH dependency . Aliphatic alcohol acceptors show small increases in acceptor activity between pH 6 and 8, with 5-fold increases from pH 8 to 10 (at pH 10, glycerol phosphates:Pi = 2.5) . 31P NMR inversion transfer has been used to measure the koff for Pi dissociation from the noncovalent enzyme complex (E . P) . For the Zn(II)4 alkaline phosphatase koff is essentially pH independent at approximately 35 s-1 . For Cd(II) or Mg(II) at the B site in place of Zn(II), koff less than or equal to 1 s-1 X Cl-ion, which appears to coordinate the A site metal ion, enhances koff, suggesting that both Cl- and HPO2-4 can coordinate the A site metal ion in a 5-coordinate intermediate . pH control of the alkaline phosphatase mechanism appears to reside in the stability of E-P and not the dissociation of E . P, compatible with the hypothesis that the activity-linked pKa is that of a H2O molecule coordinated to the A site metal, which in the hydroxide form becomes the nucleophile attacking the phosphoseryl group (E-P). J Biol Chem, 1985 Mar 10, 260(5), 2862 - 8 Channeling of a beta-oxidation intermediate on the large subunit of the fatty acid oxidation complex from Escherichia coli; Yang SY et al.; The kinetic properties of the fatty acid oxidation complex from Escherichia coli were studied with the aim of elucidating the functional consequence of having enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase associated with a multifunctional polypeptide . The kinetic parameters of individual enzymes were determined and used in model calculations based on a published theory (Storer, A . C., and Cornish-Bowden, A . (1974) Biochem . J . 141, 205-209) to predict the kinetic behavior of a system of functionally unlinked enzymes . The validity of the theory for making these calculations was proven by demonstrating a good agreement between the calculated and observed rates of intermediate and product formation for the conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by a mixture of bovine liver enoyl-CoA hydratase and pig heart L-3-hydroxyacyl-CoA dehydrogenase . The conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by the sequential action of the hydratase and dehydrogenase of the complex from E . coli was determined by measuring the rate of NADH formation . Stopped-flow measurements showed the rate of NADH formation to be linear without any lag period . When the initial velocity of the hydratase was 10.2 microM min-1, that of the overall reaction was 8.41 microM min-1 . In contrast, the results calculated by use of the Storer and Cornish-Bowden equation for a system of unlinked enzymes predicted the overall reaction to exhibit a lag time of 30 s and to result in the accumulation of 2.1 microM 3-hydroxydecanoyl-CoA before reaching a velocity corresponding to 82.5% of that of the hydratase reaction . The high initial rate and the unusual kinetic properties of the overall reaction observed in the present study are best explained by a channeling mechanism on the large subunit of the E . coli fatty acid oxidation complex . When the apparent degree of channeling is corrected for the percentage of the dehydrogenase active sites saturated with NAD+, more than 90% of the intermediate appears to be transferred directly from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase. J Biol Chem, 1985 Mar 10, 260(5), 2737 - 41 Subunit M2 of mammalian ribonucleotide reductase . Characterization of a homogeneous protein isolated from M2-overproducing mouse cells; Thelander M et al.; The M2 subunit of mammalian ribonucleotide reductase was purified to homogeneity from hydroxyurea-resistant, M2-overproducing mouse cells . The purification procedure involved affinity chromatography on an anti-tubulin antibody-Sepharose column and high performance gel permeation chromatography . The pure protein is a dimer of Mr = 88,000, containing stoichiometric amounts of a non-heme iron center and a tyrosyl free radical . The radical is destroyed by hydroxyurea but can readily be regenerated on incubation of the radical-free protein alone with iron-dithiothreitol in the presence of air . The ability to spontaneously regenerate the tyrosyl radical distinguishes protein M2 from the corresponding subunit of Escherichia coli ribonucleotide reductase, protein B2, but apart from that the two proteins are very similar. J Biol Chem, 1985 Mar 10, 260(5), 3063 - 70 Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein; Aiba H; The regulatory region of the Escherichia coli cya gene was analyzed by using S1 nuclease mapping and in vitro transcription experiments . The cya gene was transcribed, both in vivo and in vitro, from one major promoter (P2) and two weak promoters (P1 and P1') that are located about 200 base pairs upstream of P2 . The transcription from P2 was specifically inhibited by cAMP-CRP (cAMP receptor protein) in vitro . This regulatory mechanism was shown to be physiologically relevant through quantitative analyses of the cya mRNA in intact cells by S1 and dot blot assays . DNase I protection experiments revealed that cAMP-CRP binds to the cya DNA region between +11 and -20, in which a consensus CRP binding sequence is present . Moreover, it was found that cAMP-CRP alters the binding of RNA polymerase to the promoter region, thus inhibiting the transcription of the cya gene. Science, 1985 Mar 8, 227(4691), 1238 - 40 A second nuclear protein is encoded by Epstein-Barr virus in latent infection; Hennessy K et al.; A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame . Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli . Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro . This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA. Biochim Biophys Acta, 1985 Mar 8, 838(3), 335 - 42 Evidence for multiple molecular weight forms of somatostatin-like material in Escherichia coli; LeRoith D et al.; Extracts of Escherichia coli grown in defined medium contain somatostatin-related material (1-10 pg/g wet weight of cells) . Preconditioned medium had no immunoactive somatostatin whereas, conditioned medium had 110-150 pg/l . Following purification of the extracted material on Sep-pak C18, Bio-Gel P-6 and HPLC, multiple molecular weight forms of somatostatin- (SRIF-) related material were identified . The material in one peak reacted in both the N-terminal and C-terminal SRIF immunoassay and coeluted on HPLC with SRIF-28, whereas that in a second peak eluted near SRIF-14 and was reactive only in the C-terminal SRIF assay . The two peaks are thus similar to SRIF-28 and SRIF-14 of vertebrates . These findings add support to the suggestion that vertebrate-type peptide hormones and neuropeptides have early evolutionary origins. Nature, 1985 Mar 7-13, 314(6006), 67 - 73 Hypervariable 'minisatellite' regions in human DNA; Jeffreys AJ et al.; The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli . Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite . A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis. J Mol Biol, 1985 Mar 5, 182(1), 45 - 65 Mutagenic specificity of ultraviolet light; Miller JH; Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v . stimulates many types of mutations . In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5% . A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations . Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions . G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines . In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions . Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence . Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs . No additions were detected . The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1) . In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence . For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots . The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion. J Med Chem, 1985 Mar, 28(3), 303 - 11 Receptor-based design of dihydrofolate reductase inhibitors: comparison of crystallographically determined enzyme binding with enzyme affinity in a series of carboxy-substituted trimethoprim analogues; Kuyper LF et al.; By the use of molecular models of Escherichia coli dihydrofolate reductase (DHFR), analogues of trimethoprim (TMP) were designed which incorporated various 3'-carboxyalkoxy moieties in order to acquire ionic interactions with positively charged active-site residues . Certain of these compounds have shown exceptionally high affinity for this enzyme . For example, the 3'-(carboxypentyl)oxy analogue was found to be 55-fold more inhibitory than TMP toward E . coli DHFR (Ki = 0.024 nM vs . 1.32 nM for TMP) . X-ray crystallographic studies of E . coli DHFR in binary complexes with TMP and two members of this acid-containing series of compounds defined the binding of these inhibitors and showed the carboxyl group of the latter two inhibitors to be ionically bound to Arg-57 . These observations were in agreement with postulated binding modes that were based on receptor modeling. Virology, 1985 Mar, 141(2), 190 - 200 Structure of phage phi 29 connector protein assembled in vivo; Carrascosa JL et al.; The protein p10 that forms the connector of phage phi 29, has been produced in Escherichia coli harboring a plasmid that carried the gene coding for this protein . The connector protein is assembled in a 13.4-S oligomer that has an apparent molecular weight of 460,000, suggesting that it is a dodecamer . The purified oligomers have been studied by electron microscopy of the isolated particles as well as by image-processing techniques (Fourier and rotational filtering) of artificially induced two-dimensional aggregates . The results show that the purified p10 is assembled in a circular structure with a hole in its center and 12 morphological units in the periphery . Both the morphology and the dimensions of this p10 oligomer are very similar to those of the upper neck collar extracted from phi 29 viral particles . The results strongly suggest the close relationship between the p10 oligomers assembled in E . coli and the ones produced in phi 29 infected Bacillis subtilis. EMBO J, 1985 Mar, 4(3), 775 - 80 Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies; Schoemaker JM et al.; Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies . These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together . We have examined structural aspects of inclusion bodies made in E . coli as a result of high level expression of the eukaryotic protein, calf prochymosin . Prochymosin is a monomeric protein containing three disulfide bridges . It was expressed at up to 20% of cell protein from a plasmid containing the E . coli tryptophan promoter, operator and ribosome binding site . Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels . When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds . The inclusion bodies also contained a high concentration of reduced prochymosin . The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E . coli. Am J Vet Res, 1985 Mar, 46(3), 711 - 8 Effects of endotoxemia on lung water and hemodynamics in conscious calves; Olson NC et al.; The effects of endotoxemia on cardiovascular and pulmonary parameters were determined in conscious, 4- to 6-week-old calves . Escherichia coli endotoxin was infused continuously (4 micrograms/kg/hr, IV) for 5 hours . During endotoxemia, pulmonary vascular resistance and mean pulmonary artery pressure increased, and cardiac index, central plasma volume, and mean systemic arterial pressure decreased . Neutrophil, lymphocyte, and platelet counts also decreased . During the first hour of endotoxemia PaO2 decreased, and alveolar-arterial O2 gradient and shunt fraction increased . Lung extravascular thermal volume was increased from 2 to 5 hours . Postmortem extravascular lung water/extravascular dry weight ratio, bronchoalveolar lavage albumin, and poly morphonuclear cell content did not change . Microscopically, the septal capillaries of endotoxemic calves were dilated and engorged with erythrocytes, accompanied by focal accumulations of neutrophils . Intraalveolar edema and hemorrhage were not seen. J Clin Invest, 1985 Mar, 75(3), 818 - 24 Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation; Colucci M et al.; Endotoxin producing bacteria cause disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in man is still unclear . Impairment of the fibrinolytic system has been suggested as a contributing mechanism . A single injection of Escherichia coli lipopolysaccharide in rabbits resulted in a marked and prolonged increase of the levels of a fast-acting inhibitor of plasminogen activator (PA-inhibitor) in plasma (from 3.9 +/- 0.7 to 41 +/- 13.2 U/ml after 3 h) . Gel filtration studies indicated that inhibition of human tissue-type plasminogen activator (t-PA) by rabbit plasma is accompanied by a change in the elution profile of the activator compatible with the formation of an enzyme-inhibitor complex with an apparent molecular weight of 100,000 . Injection of human t-PA (1,500 IU/kg body wt) in endotoxin treated animals resulted in very fast inhibition of t-PA and formation of a similar complex . The half-life of circulating PA-inhibitor activity in rabbits was about 7 min as estimated by donor receiver plasma transfusion experiments . Stimulation of cultured human endothelial cells with endotoxin resulted in enhanced rate of accumulation of PA-inhibitor activity in the culture medium (two- to sevenfold increase) . In five patients with septicemia, markedly increased levels of PA-inhibitor (14.3 +/- 15.5 U/ml) as compared with control subjects (1.3 +/- 0.7 U/ml) were observed in plasma . A very strong correlation (r = 0.98) was found between inhibition of t-PA and of urokinase in all conditions, suggesting that this fast-acting inhibitor reacts with both plasminogen activators . These data suggest that the appearance of this fast-acting PA-inhibitor is very sensitive to endotoxin stimulation . The marked increase in the level of PA-inhibitor in blood may contribute to the pathogenesis of DIC in septicemia. Mutat Res, 1985 Mar, 149(1), 17 - 23 Bio-antimutagenic effects of tannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r; Shimoi K et al.; Tannic acid suppressed the mutagenesis in E . coli B/r WP2 trp- induced by UV or 4-nitroquinoline 1-oxide (4NQO), but not that induced by gamma-rays or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The depression of mutations induced by UV was most remarkable in the DNA-repair-proficient strain (WP2) . Tannic acid, however, showed no bio-antimutagenic effect in the excision repair-deficient strain (WP2s uvrA- or ZA159 uvrB-) under the test conditions where no cellular toxicity was observed . The effect ceased within 30 min after UV irradiation . The inhibition of the expression of Trp+ phenotype and the delay of the first cell division after UV irradiation were not observed in the presence of tannic acid . From these results we conclude that tannic acid may enhance the excision-repair system probably by activating the repair enzymes or by interacting with DNA. Mol Gen Mikrobiol Virusol, 1985 Mar, (3), 15 - 9 {Characteristics of replication of small colicinogenic plasmids}; Zverev VV et al.; Specificity of small multicopy colicinogenic plasmids ColA, ColD, ColE2 and ColK replication has been compared with the one of ColE1 plasmid . Copy number for these plasmids per host cell has been estimated under the normal conditions of cellular growth and under the conditions of chloramphenicol-inhibited growth . DNA polymerase I and dnaB protein, an obligatory component for elongation step in replication, have been shown to be necessary for the plasmids replication . Initiation of plasmids replication has been demonstrated to be independent of dnaA and dnaC proteins . Replication of plasmid ColE2, being similar in its main features to replication of other plasmids from this group, has an important distinction . It requires de novo protein synthesis implying that ColE2 replicon may be different from ColA, ColD, ColK, ColE1 replicons . Thus study of the inducible A, D, K, El colicin synthesis coded by the corresponding plasmids has revealed the similarity regulation of genes, determining the synthesis of each of the mentioned colicins. Z Gastroenterol, 1985 Mar, 23(3), 130 - 8 {Protease inhibitors, serum endotoxin and serum immunoglobulins following portacaval end-to-side anastomosis in animal experiments}; Machraoui A et al.; In order to clarify the pathophysiological mechanism of certain biochemical and immunological changes (endotoxin in serum, protease inhibitors, immunoglobulins) found in a former study on human cirrhosis of the liver the porto-caval end-to-side anastomosis of rats with unaffected livers was chosen as test model . With the aid of this bypass the "spill-over" phenomenon of the liver can be completely imitated . In this study, 7 operated and 5 or 14 control animals resp . are referred to . Serum endotoxin, acid-stable and acid-unstable protease inhibitors and immunoglobulins IgG, IgA and IgM were determined 20 months after operation . For the determination of potential hemodynamically or toxically induced effects on these organs, morphologic liver and lung examinations were performed . On the average, the operated rats showed a weight loss of 8 percent, i.e . from 378.4 +/- 9.2 g to 348.4 +/- 17 g . Compared to control rats, their relative liver weights were significantly lower (34%) (mean = 2.23 +/- 0.2 compared to 3.4 +/- 0.44 g, p less than 0.0005) . Serum immunoglobulins IgG, IgA and IgM in operated animals were significantly higher (p less than 0.005 or p less than 0.025 resp . and 0.0025) . Endotoxin in serum could be identified in 4 out of 7 operated animals (57, 1%), but in none of the control animals . While there was no difference in serum levels of acid-unstable protease inhibitors between the two groups levels of acid-stable protease inhibitors were in operated animals by 24% higher than in control animals (mean = 35.3 +/- 3.3 compared to 28.5 +/- 2.3 mU/ml, p less than 0.0005).(ABSTRACT TRUNCATED AT 250 WORDS) Biochimie, 1985 Mar-Apr, 67(3-4), 353 - 6 Role of Escherichia coli RecA protein in SOS induction and post-replication repair; Moreau PL; The RecA protein of Escherichia coli plays a central role in DNA repair mechanisms . When it is incubated with single-stranded DNA and a nucleoside triphosphate, the purified RecA protein acts both by promoting cleavage of the LexA protein, the repressor of the SOS genes, and by catalyzing strand exchange between a variety of DNA molecules . A model for the regulation of the activity of the RecA protein in a cell exposed to a DNA damaging treatment is proposed. Biochimie, 1985 Mar-Apr, 67(3-4), 349 - 52 RECA immunological assay as a tool to analyze the SOS response; Salles B et al.; The content of RECA protein, one of the SOS genes product, was determined in a bacterial extract by a two site-radioimmunometric assay . The variation of the RECA concentration after induction by physical or chemical treatments was used as a probe to analyze the SOS response . Relationships between either the number or the nature of DNA lesions and the level of the relative amplification of RECA have been established . The modulation of the recA gene expression is discussed. J Biochem (Tokyo), 1985 Mar, 97(3), 851 - 7 Active site-directed modification of tryptophanase by 3-bromopyruvate; Honda T et al.; Tryptophanase purified from Escherichia coli B/It7-A was irreversibly inactivated by 3-bromopyruvate following pseudo-first-order kinetics . The inactivation rate for the holoenzyme tended to saturate as the concentration f bromopyruvate increased . L-Alanine and DL-3-phenylserine, potent competitive inhibitors with respect to L-tryptophan decomposition, protected the enzyme from inactivation . Titration of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of one SH group per enzyme subunit resulted in a complete inactivation . When the enzyme was subjected to bromopyruvate-modification following pretreatment with DTNB, the activity was almost completely restored upon reduction with dithiothreitol . Modification of the enzyme with bromopyruvate quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton . These results support the possibility that bromopyruvate reacts with the enzyme as an affinity labeling agent. Mikrobiologiia, 1985 Mar-Apr, 54(2), 252 - 6 {Effect of acetate on the growth of Escherichia coli during aerobiosis and anaerobiosis}; Smirnova GV et al.; The dynamics of acetate accumulation was studied in Escherichia coli K-12 batch cultures with a substrate addition . At pH 7.0, the growth stopped at 150 mmoles of acetate per litre of the medium under the aerobic conditions or at 35 mmoles of acetate per litre of the medium under the anaerobic conditions . Experiments with extraneous addition of acetate suggest that acetic acid plays a key role in inhibiting the growth of E . coli by acid metabolites . The authors propose a hypothetical mechanism to account for the inhibiting action of acetate. Mikrobiologiia, 1985 Mar-Apr, 54(2), 227 - 32 {Stability and dynamics of R-plasmids in Escherichia coli populations in continuous cultivation}; Filonov AE et al.; The stability of the conjugative plasmid RP4 and the nonconjugative plasmid pBS94 in Escherichia coli C600 cells containing both plasmids was studied in continuous cultivation under chemostat and pH-stat conditions . The plasmids remained stable in the cells of the bacterial population for 100 generations, and no cells were found without the plasmids . The competition between strains with and without the plasmids in a mixed culture resulted in the removal of the plasmid-free strain from the population . In these experiments, conjugative transfer of plasmids into the plasmid-free strain was observed, and co-transfer of both plasmids was more effective under the pH-stat conditions. EMBO J, 1985 Mar, 4(3), 829 - 35 The solution structure of a B-DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon . Refinement on the basis of interproton distance data; Clore GM et al.; A restrained least squares refinement of the solution structure of the double-stranded DNA undecamer 5'd(AAGTGT-GACAT).5'd(ATGTCACACTT) comprising a portion of the specific target site of the cAMP receptor protein in the gal operon is presented . The structure is refined on the basis of both distance and planarity restraints, 2331 in all . The distance restraints comprise 150 interproton distances determined from pre-steady state nuclear Overhauser enhancement measurements and 2159 other interatomic distances derived from idealized geometry (i.e., distances between covalently bonded atoms, between atoms defining fixed bond angles, and between atoms defining hydrogen bonding in AT and GC base pairs) . Two refinements were carried out and in both cases the final RMS difference between the experimental and calculated interproton distances was 0.2 A . The difference between the two refined structures is small (overall RMS difference of 0.23 A) and represents the error in the refined coordinates . Although the refined structures have an overall B-type conformation there are large variations in many of the local conformational parameters including backbone and glycosidic bond torsion angles, helical twist and propellor twist, base roll and base tilt angles. Ann Inst Pasteur Microbiol, 1985 Mar-Apr, 136A(2), 203 - 12 {Comparative study of adherence to rabbit enterocytes, presence of colonization factors CFA/I and CFA/II and toxinogenesis of 55 strains of Escherichia coli}; Germani Y et al.; Fifty-five strains of Escherichia coli isolated from 51 faeces of Melanesian children with acute diarrhoea in New Caledonia were studied; three diarrhoeas were bloody . For each strain, haemagglutination type, adhesion to rabbit enterocytes, serotype, production of heat-labile (LT) or heat-stable (ST) toxins and identification of colonization factor antigens CFA/I or CFA/II were determined . We identified 48 strains able to attach to rabbit enterocytes; 27 produced enterotoxins (21 strains LT+ and 6 ST+) and 19 had CFA (13 CFA/I and 6 CFA/II) . Five serotypes were identified: O6, O78, O80, O114 and O127:B8 . One strain, O127:B8, which was able to attach to enterocytes, had CFA/I and produced LT toxin. Plasmid, 1985 Mar, 13(2), 81 - 7 Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1; Nordstrom M et al.; The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical . Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number . The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively . These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid . Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific . These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
